Analytical and Isolation Methods

Before 1970, fractional vacuum distillation, analytical and preparative chromatographic procedures such as column chromatography (CC), TLC, GC, and chemical methods were used. These procedures have been extended to HPLC, high-performance gas chromatography (HPGC) (associated or not associated with specific detectors), direct vaporization in the GC apparatus, and dynamic headspace techniques.

Liquid Column Chromatography

Liquid column chromatography is the oldest method used in organic chemistry since Tswett's discovery in 1906. Essential oils from ginger can be fractionated on a silica gel column into a hydrocarbon fraction and an oxygenated fraction. Elution solvents are nonpolar hydrocarbons such as pentane, and for the latter a mixture of pentane with ether (2:1, v/v) or acetone (4:1, v/v), respectively (Van Beek, 1991). Thus essential oils with removed terpenes can be obtained on a large scale. However, terpenes containing a furan ring such as perillene and rosefuran found in ginger oil occur in both fractions. More polar compounds such as aliphatic acids can be eluted with a more polar solvent (pentane/ethanol: 9:1 v/v). Chromatographic procedures on alumina were used by Herout et al. (1953) for the isolation of some sesquiterpene hydrocarbons: ( + )-ar-curcumene, bisabolene, farnesene, and a-zingiberene. But this class of compounds was better separated by column chromatography on silver nitrate—treated supports (Smith and Ohlson, 1962). Balladin and Headley (1999) isolated essential oil and pungent principles of West Indian ginger by liquid chromatography using silica gel (70 to 230 mesh) and a mixture of petroleum ether (60 to 80° C) and diethyl ether (3:7 v/v) as the mobile phase. They isolated seven fractions: the first 15 mL contained the very volatile and less polar compounds present in the extracted oleoresin from the sun-dried ginger rhizome; that is, the essential oil accounts for 25.6% (wt/wt) of the total oleoresin charge to the column. The next 5 mL aliquot was without any compound. The following 25 mL contained the shogaol fraction and represented 47.3% of the sample. The next 5 mL aliquot was without any compound. The following 35 mL contained the gingerol fraction and represented 27.1% of the sample. Each fraction was subjected to GC/MS and HPLC/MS analyses. Two techniques of column chromatography have been used by Zarate et al. (1992) to separate pungent principles of ginger: vacuum and flash chromatography, with toluene/methanol (16:10 v/v) as the mobile phase. Whereas flash chromatography is not very successful for separation of these compounds, vacuum chromatography is more rapid and effective.

Purification and characterization of cysteine proteinase from fresh ginger rhizome has been carried out by column chromatography using diethylaminoethyl (DEAE)-cellulose and Sephadex G 75 (Kitamura and Naguno, 2000). Two CPI fractions of 11,000 to 11,800 and 15,500 to 16,000 molecular weights were recorded. Both fractions showed potent papain inhibitory activities and were stable at <40 to 60° C, but the activities decreased and disappeared when exposed to higher temperatures.

Thin-Layer Chromatography

Since the time Stahl published his work (1962), TLC, a fast, easy, and inexpensive method, has been widely used in organic chemistry (Vernin, 1970). Unfortunately, the method is unsuitable for complex mixture analysis such as essential oils. However, it is adequate for the preparative separation of some compounds or a set of compounds having the same retention times (Rt). The extract after solvent extraction can be submitted to GC and GC/MS analyses. Quantitative determination by ultraviolet (UV) densitometry can also be used in a simple case. Analyses for the ginger oleoresin have been reported by Connell (1970) using TLC on silica gel plates with hexane-diethyl ether mixtures as eluent. Quantitative determination by densitometry allowed him to separate three main groups: gingerols, shogaols essential oils, and more polar and heavy compounds as a trailing. Some sesquiterpene hydrocarbons were separated by TLC on silica gel plates treated with silver nitrate (Connell, 1970). Analysis of gingerol compounds of raw ginger and its paste was carried out by TLC (Jo, 2000). Antioxidant compounds in ginger rhizomes from Korea, extracted with ethyl acetate from crude methanol extract, were separated through TLC. Ten phenolic antioxidant bands were visualized through color reactions using ferric chloride, potassium ferrocyanide, and 1,1-diphenyl-2-picrylhydrazyl (DPPH) and were purified through preparative TLC and HPLC. Among them, five antioxidants were identified as (4)-, (6)-, and (10)-gingerols and (6)-shogaol on the basis of their molecular weight determination through LC/MS. As shown in experiments using DPPH free radicals, (6)-gingerol and PT4-HPS were revealed to be more efficient than BHT (butylated hydroxytoluene). Total gingerol content (determined through reversed phase HPLC) in rhizomes of different ginger varieties varied significantly. Two varieties collected in Korea (HG 55) and in Brazil (HG 52) showed the highest content.

High-Performance Liquid Chromatography

HPLC has supplanted the TLC for the preparative separation of essential oil compounds and for the quantitative determination of important and heavy compounds. Two examples have been given for ginger oil by Van Beek (1991). The first example concerns the separation of the more important sesquiterpene hydrocarbons of ginger oil from India, accounting for 70% of the oil. The following conditions were used: a 25 X 1 cm column fitted with 5^m C-18 silica gel reversed phase eluted with MeCN/H2O (88:12) with a flow rate of 4 mL/min, and UV detection at 215 and 245 nm. After the four preparative runs, ar-curcumene was obtained in >99% purity and (E,E)-a-farnesene in 84% purity. Other sesquiterpene hydrocarbons (P-sesquiphellandrene, a-zingiberene, P-bisabolene) have been separated under different analytical conditions. Two fractions were collected. The first consisted of 53% a-zingiberene, 19% P-bisabolene, and 9% P-sesquiphellan-drene. They were further purified by means of preparative capillary GC. Using a reversed phase system: HPLC column 15 X 0.46 cm fitted with Microsorb 5 ^m C-18 silica gel, solvent: MeCN/H2O (6/1, 1 mL.min-1) to MeCN/H2O (0.5/5 in 30 minutes) and a detection UV at 236 nm, the geranial and neral content of any ginger oil can be measured in minutes (the minimum detectable quantity was 1 ng) (Van Beek, 1991). Other compounds detected were: myrcene, ß-phellandrene, (E,E)-a-farnesene, and ß-sesquiphel-landrene + a-zingiberene. The comparison by HPLC of the extracts of Indian ginger root obtained by the conventional methods and that obtained by supercritical CO2 shows that steam distillation is not suitable for the extraction of the pungent principles of ginger oleoresin. The supercritical CO2 method gives better results than the hexane extracts (see Table 3.7) (Pellerin, 1991). Paradol has not been taken into account.

A quantitative method by HPLC of pungent principles of ginger was developed by Yoshikawa et al. (1994). The content of (6)-, (8)-, and (10)-gingerols, (6)- and (8)-shogaols, 6-dihydrogingerdione, and galanolactone in 20 kinds of rhizomes originating from China, Taiwan, Vietnam, and Japan, and fresh ginger root cultivated in Shizuoka Prefecture of Japan were examined. It was found that Japanese and fresh ginger root contained gingerols, shogaols, 6-dehydrogingerdione, and galanolactone as the major constituents.

A HPLC method was developed by Sane et al. (1998) to study the geographical variation in the ginger samples obtained from different states of India with respect to their gingerol and ginger oil content. Analyses of gingerol compounds of raw ginger and its paste were carried out by a combination of TLC and HPLC with Licrosorb RP-18 column by Jo (2000). (6)-, (8)-, and (10)-Gingerols were identified by HPLC/MS, and nuclear magnetic resonance (NMR). The content of (6)-, (8)-, and (10)-gingerols were 635.3, 206.3, and 145.7 mg %, respectively, in raw ginger (from Korea). They were 418.2, 142.6, and 103.3 mg % in ginger paste, respectively. Another HPLC method was applied by Chen et al. (2001) for the determination of pungent constituents in ginger and to evaluate ginger extracts obtained by supercritical CO2 or anhydrous alcohol extraction. The effective content of pungent constituents was 13.84 and 1.46%, respec-

Table 3.7 HPLC comparison of gingerols and shogaols obtained from Indian ginger extracts by three different extraction methods*

Percentages (%)

Table 3.7 HPLC comparison of gingerols and shogaols obtained from Indian ginger extracts by three different extraction methods*

Percentages (%)

Pungent principle**

Steam distillation

Supercritical CO2

Hexane

(6)-Gingerol

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