ESSENTIAL OIL OF GINGER AND ITS OLEORESIN 1 FLAVOUR AND FRAGRANCE JOURNAL Flavour Fragr. J.

2005; 20: 16 Published online 27 October 2004 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/ffj.1373

Studies on essential oils, Part 42: chemical, antifungal, antioxidant and sprout suppressant studies on ginger essential oil and its oleoresin
Gurdip Singh,1* Sumitra Maurya,1 C. Catalan2 and M. P. de Lampasona2
1 2

Chemistry Department, D.D.U. Gorakhpur University, Gorakhpur-273009, India Instituto de Quimica, Organica Universidad Nacinal de Tucuman, Ayacucho 471, S.M. de Tucuman 4000, Argentina

Received 29 April 2003; Revised 24 October 2003; Accepted 13 November 2003

ABSTRACT: GCMS analysis of fresh rhizome essential oil of Zingiber ofcinale showed the presence of 69 components, accounting for 96.93% of the total oil. The major component was -zingiberene (28.62%) followed by camphene (9.32%), ar-curcumene (9.09%) and -phellandrene (7.97%). Analysis of the oleoresin showed the presence of 34 components, accounting for 88.63% of the total oleoresin. The major components were trans-6-shogaol (26.23%), trans-10shogaol (13.0%), -zingiberene (9.66%) and 10-gingerdione (6.80%). Moreover, the essential oil was found to be 100% antifungal against Fusarium oxysporum, whereas the oleoresin was 100% antifungal against Aspergillus niger. The latter expressed better antioxidant activity in sunower oil as compared to the essential oil and synthetic antioxidants (BHA and BHT). Copyright 2004 John Wiley & Sons, Ltd. KEY WORDS: Zingiber ofcinale; antioxidant; sprout suppressant studies; peroxide value

Introduction
Ginger is the dried rhizome of Zingiber ofcinale Roscoe (Family Zingiberaceae), a herbaceous perennial species. The country of origin of the plant is not known with certainty, but it is presumed to be in the region of India or China. The knotted and branched rhizome, commonly called the root is the portion of ginger used for culinary and medicinal purposes. It is extensively used in the formation of compounded aromas for avouring confectionery, bakery products, condiments, sauces and carbonated beverages. The dried powder is used as a spice. Extracts and oleoresins are produced from dried and non-peeled ginger, since peeled ginger losses much of its essential oil content.2 The oleoresin contains the essential oil and non-volatile pungent principles. It represents the total avouring component of spices in highly concentrated form and is very close to the whole ground ginger. Lipid oxidation is a major cause for the deterioration of fat-containing food. It initiates other undesirable changes in food, affecting its nutritional quality, colour, avour and texture. Auto-oxidation of polyunsaturated lipids involves a free radical chain reaction, generally initiated by exposure of the lipids to light, heat, ionizing

radiation, metal ions or metaloprotein catalysts. Therefore, the inhibition of free radical oxidation by antioxidants is of great practical importance in preserving polyunsaturated lipids from deterioration. Synthetic compounds, such as Butylated hydroxyl toluene (BHT) and Butylated hydroxyl anisole (BHA), are widely used antioxidants due to their low cost, high stability and effectiveness. However, due to possible toxicological side-effects of synthetic antioxidants on human health, the general trend towards reducing the use of synthetic food additives has resulted in an expansion of the search for natural substances possessing antioxidative properties. For this purposes, essential oils and oleoresins are widely used globally. Very little work has been reported on the chemistry37 and activity 812 of the essential oil and oleoresin of the ginger plant. In continuation of our research programme1318 on the essential oils, chemical, antifungal, antioxidant and sprout-suppressant studies of fresh ginger rhizome essential oil and its oleoresin have been undertaken and the results are reported in this communication.

Materials and Methods

Plant material

* Correspondence to: G. Singh, Chemistry Department, D.D.U. Gorakhpur University, Gorakhpur-273009, India. E-mail: gsingh4us@yahoo.com Contract/grant sponsor: Life Sciences Research Board, DRDO, New Delhi, India.

Fresh rhizomes of ginger were purchased from the local market of Gorakhpur and voucher specimens were deposited at the Herbarium of the Science Faculty of DDU Gorakhpur University, Gorakhpur.

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2 G. SINGH ET AL.

Isolation of the Oil The nely sliced rhizomes were hydrodistilled in a Clevenger-type apparatus for 6 h. A yellow-coloured essential oil (yield 1.2%) was obtained. It was dried over anhydrous sodium sulphate to remove traces of moisture.

from the GCMS percentage of total ion current peak. Chemical constituents were identied by comparing their mass spectra with library data19,20 from NBS 75K and/or by co-injection with authentic samples. The results for the essential oil and its oleoresin are reported in Tables 1 and 2, respectively.

Isolation of the Oleoresin After the extraction of the essential oil, the sliced rhizomes were dried and then, utilizing a Soxhlet apparatus, oleoresin was obtained using acetone as a solvent.

Antifungal Investigations The antifungal activity of the essential oil and its oleoresin against the pathogenic fungi Aspergillus terrus (AT), Aspergillus niger (AN), Aspergillus avus (AF), Trichothecium roseum (TR), Fusarium graminearum (FG), Fusarium oxysporum (FO), Fusarium monoliforme (FM) and Curvularia palliscens (CP) was conrmed using the food poison and inverted Petri-plate technique.21 These fungi were collected from the Microbial Type Culture Collection (MTCC), Chandigarh, India. The cultures were maintained in oatmeal agar medium containing oatmeal 60 g/l and agar 20 g/l, adjusting the pH to 6.06.5. The required dose (2, 6 and 10 l) of the oil and oleoresin were mixed with the culture medium. Each test was replicated three times and fungal toxicity was calculated in terms of percentage mycelial inhibition, using the following formula: % mycelial zone inhibition =

Chemical Investigation Chemical analysis of the essential oil and its oleoresin was undertaken by gas chromatography (GC) and gas chromatographymass spectroscopy (GCMS) techniques. GC Using a Hewlett-Packard 5890 Series II gas chromatograph equipped with ame ionization detector (FID) and silica column, a gas chromatogram of the essential oil and its oleoresin were obtained. The column was an HP5 (30 m 0.32 mm i.d.); injector and detector temperatures were maintained at 250 C and 300 C, respectively. The amount of the samples injected was 0.1 l (in split mode). Helium was used as carrier gas at a ow rate 1.0 ml/min. The oven temperature was programmed as follows: 65 for 1 min, rising at 1 C/min to 75 C, held for 2 min and raised at 0.5 C/min to 81 C, held for 2 min, again raised at 3 C/min to 180 C, then held for 7 min. GCMS The essential oil and its oleoresin were subjected to GCMS analysis using a Hewlett-Packard mass detector (Model 5973) and a HP-5 MS column (30 m 0.25 mm i.d., lm thickness 0.25 m). The injector, GCMS interphase, ion source and selective mass detector temperatures were maintained at 270 C, 280 C, 230 C and 150 C, respectively. The oven temperature for essential oil and its oleoresin was programmed as follows: 60 C for 1 min rising at 1.5 C/min to 185 C and hold for 1 min, then rising at 9 C/min to 275 C, then hold for 2 min.

dc dt 100 dc

where dc and dt are average diameters of the mycelial colonies of the control and treated sets, respectively. The results are given in Tables 3 and 4.

Antioxidant Activity Rened sunower oil without any additive was obtained from Custom Laboratory, New Custom House, Mumbai, as a gift sample. The oil was selected for the present investigation due to its high degree of unsaturation and general use as an edible oil. Essential oil, oleoresin, BHT and BHA were mixed with sunower oil at 200 ppm concentration. A control sample of sunower oil was also prepared under similar conditions. All samples were incubated at 70 C in 100 ml uncovered beakers.

Peroxide Value The peroxide value of all the samples was determined22 after 7, 14, 21 and 28 days. For this purpose, a known weight of sample (1 g) was dissolved in a mixture of CH3COOH:CHCl3 (3:2 v/v) and then a saturated solution of potassium iodide (1 ml) was added. The liberated I2 was titrated against sodium thiosulphate (0.1 N), using

Identication of Components The percentage of components was taken from capillary GC traces with FID wherever available or directly

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ESSENTIAL OIL OF GINGER AND ITS OLEORESIN 3

Table 1. Chemical composition of Zingiber ofcinale essential oil Compound

2-Methyl-3-buten-2-ol Hexanal 2-Heptanone Tricyclene -Thujene -Pinene Camphene Sabinene -Pinene 6-Methyl-5-hepten-2-one Myrcene n-Octanal -Phellandrene -3-Carene -Terpinene p-Cymene -Phellandrene 1,8-Cineole (Z)- -Ocimene (E)- -Ocimene -Terpinene cis-Sabinene hydrate n-Octanol Terpinolene Linalool Nonanal cis-p-Menth-2-en-1-ol Camphor Camphene hydrate Citronellal endo-Borneol Terpinen-4-ol -Terpineol Decanal Nerol Citronellol Neral Geraniol Geranial endo-Bornyl acetate 2-Undecanone -Elemene Cyclosativene -Copaene -Elemene -Caryophyllene -Elemene trans- -Bergamotene Aromadendrene trans- -Farnesene -Gurjunene Germacrene D ar-Curcumene Valencene -Zingiberene -Bisabolene E,E- -Farnesene -Cadinene -Sesquiphellandrene trans- -Bisabolene Elemol Germacrene B trans-Nerolidol trans-Sesquisabinene hydrate 10-epi- -Eudesmol Zingiberenol Binesol (agarospirol) -Eudesmol -Eudesmol Total

Table 2. Chemical composition of Zingiber ofcinale oleoresin Compounds

Camphene -Phellandrene -Elemene Cyclosativene -Copaene -Elemene -Caryophyllene -Elemene trans- -Bergamotene Aromadendrene trans- -Farnesene Germacrene-D ar-Curcumene Valencene -Zingiberene -Bisabolene E,E--Farnesene -Cadinene -Sesquiphellandrene trans- -Bisabolene Elemol Germacrene-B Zingerone cis-6-Shogaol 6-Paradol trans-6-Shogaol 6-Gingerol cis-8-Shogaol trans-8-Shogaol 6-Gingerdiol-diacetate 8-Gingerdione cis-10-Shogaol trans-10-Shogaol 10-Gingerdione Total

Area (%)
0.01 0.11 0.03 0.14 tr 2.57 9.32 0.11 0.14 0.21 0.75 0.12 0.41 0.03 tr 0.08 7.97 1.20 tr tr tr tr tr 0.15 0.47 tr tr 0.15 0.08 0.10 2.04 0.13 0.35 0.06 0.22 0.39 1.72 0.50 2.08 0.35 0.01 0.07 0.13 0.26 0.48 tr 0.07 0.07 0.11 0.12 0.11 1.03 9.09 1.42 28.62 5.40 5.52 0.17 8.64 0.38 0.49 0.43 0.47 0.40 0.06 0.92 0.11 0.25 0.11 96.93

RIa
801 890 928 931 941 953 975 980 984 993 1001 1007 1013 1020 1026 1030 1035 1040 1050 1064 1069 1072 1088 1099 1104 1124 1144 1150 1156 1167 1177 1189 1206 1230 1230 1244 1253 1273 1288 1294 1340 1370 1379 1395 1420 1433 1437 1440 1460 1475 1480 1482 1493 1495 1508 1510 1524 1526 1535 1550 1557 1564 1581

Area (%)
0.01 0.01 0.03 0.04 0.10 0.15 tr 0.04 0.04 0.04 tr 0.14 2.76 0.51 9.66 1.98 1.63 0.08 2.94 0.08 tr 0.06 0.04 3.31 1.50 26.32 1.87 1.21 7.72 2.00 1.45 3.11 13.00 6.80 88.63

tr, trace, <0.01%. Quantitative data were obtained by relating individual peak areas to the total area of the total ion chromatogram. Calibration factors were neglected.

starch as an indicator. A blank titration was also run parallel to treated samples and the peroxide value (Meq/kg) was calculated using the formula: Peroxide value (Meq/kg) = 1000SN/W where S = volume (ml) of sodium thiosulphate used (blank corrected), N = normality of sodium thiosulphate used (viz. 0.01 N) and W = weight of sample (g). The antioxidant activity of essential oil and its oleoresin in sunower oil is given in Table 5.

Sprout Suppressant Activity (SSA) Potatoes were purchased from a local market. Cotton swabs were soaked in 0.1 ml essential oil and a corresponding amount of oleoresin, dissolved in acetone and wrapped in sterilized muslin cloth. The swabs were placed at the bottom of 12 10 cm2 plastic vials and ve potatoes were taken in each vial. All vials were

tr, trace, <0.01%. Quantitative data were obtained by relating individual peak areas to the total area of the total ion chromatogram. Calibration factors were neglected. a Retention indices.

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4 G. SINGH ET AL.

Table 3. Investigations of antifungal activity of Zingiber ofcinale essential oil and its oleoresin using food poison technique Fungus Mycelial zone inhibition (%) at different doses of oil* Z. ofcinale essential oil 2 l
Aspergillus terrus (AT) Aspergillus niger (AN) Aspergillus avus (AF) Trichothecium roseum (TR) Fusarium graminearum (FG) Fusarium oxysporum (FO) Fusarium monoliforme (FM) Curvularia palliscens (CP) * Average of three replicates. 12.5 12.5 37.5 6.3 25.0 25.0 12.5 18.8

Z. ofcinale oleoresin 10 l
50.0 31.3 87.5 100.0 62.5 87.5 75.0 87.5

6 l
25.0 25.0 50.0 50.0 37.5 37.5 25.0 50.0

2 l
12.5 12.5 6.3 6.3 12.5 12.5 12.5 6.25

6 l
18.5 18.5 25.0 25.0 18.5 50.0 18.8 18.8

10 l
37.5 37.5 75.5 87.5 87.5 100.0 56.3 37.5

Table 4. Investigations of antifungal activity of Zingiber ofcinale essential and its oleoresin using inverted petriplate method Fungus Mycelial zone inhibition (%) at different doses of oil* Z. ofcinale essential oil 2 l
Aspergillus terrus (AT) Aspergillus niger (AN) Aspergillus avus (AF) Trichothecium roseum (TR) Fusarium graminearum (FG) Fusarium oxysporum (FO) Fusarium monoliforme (FM) Curvularia palliscens (CP) * Average of three replicates. 25.0 12.5 28.6 28.6 14.2 25.0 14.3 25.0

Z. ofcinale oleoresin 10 l
75.0 37.5 71.4 71.4 42.8 50.0 85.7 75.0

6 l
50.0 25.0 42.8 42.8 21.4 43.7 21.4 50.0

2 l
12.5 12.5 7.2 0.0 0.0 7.2 0.0 12.5

6 l
25.0 18.7 14.3 7.2 7.2 25.0 7.2 18.7

10 l
37.5 31.5 35.7 35.7 14.3 31.3 28.6 25.0

Table 5. Antioxidant activity of Zingiber ofcinale essential oil and its oleoresin in sunower oil at 70 C No. Additive Peroxide value* (in Meq/kg) after days 7
1 2 3 4 5 Z. ofcinale oil Z. ofcinale oleoresin Control BHA BHT 65 61 64 63 63

perforated for aeration. A control experiment was run parallel using a blank sterilized cotton swab. A weak SSA was observed every week at room temperature and average values from three experimental sets are reported in Table 6.

14
117 99 118 111 105

21
127 105 129 119 115

28
153 130 199 141 149

Results and Discussion

GC and GCMS analyses of fresh rhizome essential oil and its oleoresin were undertaken using the HP-5 series described above. The essential oil showed the

* Average of three experiments.

Table 6. Sprout inhibition activity (%) of Zingiber ofcinale essential oil and its oleoresin on potatoes Tests 1
Essential oil Oleoresin Control 80 100 0

Sprout inhibition activity (%) after weeks* 2

70 70 0

3
70 20 0

4
70 0 0

5
70 0 0

6
70 0 0

7
50 0 0

8
50 0 0

9
50 0 0

10
50 0 0

* Average of three experiments.

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ESSENTIAL OIL OF GINGER AND ITS OLEORESIN 5

presence of 69 components (Table 1), accounting for 96.93% of the total oil. The major component was zingiberene (28.62%), followed by camphene (9.32%), ar-curcumene (9.09%), -sesquiphellandrene (8.64%), -phellandrene (7.97%), E,E--farnesene (5.52%), bisabolene (5.40%), -pinene (2.57%), geranial (2.08%), endo-borneol (2.04%), neral (1.72%), valencene (1.42%), 1,8-cineol (1.20%) and germacrene D (1.03%). The oleoresin showed the presence of 34 components (Table 2), accounting for 88.63% of the total oil. The major components were trans-6-shogaol (26.32%), trans-10-shogaol (13.0%), -zingiberene (9.66%), trans-8-shogaol (7.72%), 10-gingerdione (6.80%), cis-6-shogoal (3.31%), sesquiphellandrene (2.94%), ar-curcumene (2.76%), 6gingerdiol diacetate (2.00%), -bisabolene (1.98%), 6-gingerol (1.87%), E,E--farnesene (1.63%), 6-paradiol (1.50%) and cis-8-shogaol (1.21%). Menut et al.23 reported that the essential oil obtained from this species contained citral (34.6%) and 1,8-cineol (10.4%) as the main constituents. Recently, Aggrawal et al.8 reported that the fresh rhizomes of ginger contained curcumene as the major constituent. The thermolabile zingiberene was obtained in high amount from its diethyl extract. Nishimura3 identied various components having high dilution factor, such as linalool, geraniol, neral, isoborneol, borneol, 1,8-cineol, 2-pinene-5-ol, geranyl acetate, (E)-2-octenal, (E)-2-decenal and (E)-2dodecenal, as well as (E)-2-alkanes and 2-octyl acetate, 2-pinen 5-ol, 2-(2,3-epoxy,3methyl butyl,3-methyl) furan, and (E) and (Z)-3,7-dimethyl-3,6-octadienal. Obviously, there are major differences in chemical composition between samples of essential oils obtained from this species, which may be due to genetic, environmental, developmental or other factors. The chemical composition of the oleoresin also depends on the nature of solvents used for extraction. Antifungal investigations of the oil and its oleoresin were undertaken using both the food poison (Table 3) and the inverted Petri-plate technique (Table 4). Using the food poison technique, the oil was found to be 100% antifungal against TR at 10 l dose of the oil. It was highly effective in controlling the mycelial growth of FG, AF and CP but was least effective against AN, even at a 10 L dose of the oil. The oleoresin was found to be 100% antifungal against FG. It was also highly effective in the case of FO and TR but least effective in the case of AT, AN and CP. Moreover, using the inverted Petriplate technique, the essential oil was found to be more active against AT, FM and CP; more than 75% mycelial zone inhibition was obtained: whereas the oleoresin was found to be less or ineffective as it has no vapour action. In 2001, Singh10 reported that the essential oil of ginger was known to show antifungal activity against Aspergillus species. In another study,4 dehydrozingerone imparts maximum antifungal activity among the components of ginger oleoresin.

The antioxidant activity24,25 of ginger oil and its oleoresin has been tested on sunower oil. It was observed that the oleoresin possesses better antioxidant activity than the essential oil and synthetic antioxidants (Table 5). The extent of the inhibitory effects of the oil can be attributed to the presence of an aromatic nucleus containing a polar group. The effectiveness of added materials in protecting sunower oil are in the sequence: oleoresin > BHA > BHT > oil > control. Recently, Simandi et al.26 worked on the antioxidant activity of thyme in sunower oil, using extracts of ethyl alcohol and supercritical CO2. The effectiveness of both extracts added at a level of 0.6% were equal to that of 0.1% of BHT. In sprout-suppressant studies (Table 6), the activity of oil and its oleoresin was studied using the previously described method and it was found that in the vials containing oil 50% sprout inhibition occurred, whereas in the vials containing oleoresin, 100% sprout germination occurred, hence it is concluded that the oil exhibited better activity than the oleoresin due to its vapour action. Summarizing these results, it may be concluded that the essential oil, rich in zingiberene, possesses antifungal activity against Trichothecium roseum. Its oleoresin is rich in trans-6-shogaol and is a potent antioxidant for sunower oil. The oleoresin is a potent antifungal against Fusarium graminearum.
AcknowledgementsThe authors are grateful to the Head, Prof. N.B. Singh, Chemistry Department, DDU Gorakhpur University, Gorakhpur, for providing laboratory facilities. Thanks are also due to Life Sciences Research Board, Defence Research and Development Organisation, New Delhi, for nancial assistance.

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