Antifungal Activity and Spectra Profiles of Essential Oil from Three Ginger Cultivar Rhizhomes Mangestuti, Neny Purwitasari

Department of Pharmacognocy and Phytochemistry, Faculty of Pharmacy, Airlangga University, Surabaya, Indonesia In Indonesia, three types of Ginger (Zingiber officinale Roscoe) have been distinguished (1) Jahe gajah, jahe putih besar or jahe badak (2) Jahe merah or jahe sunti (3) jahe putih kecil or jahe emprit. Their rhizomes differ in shape, colour, aroma and chemical composition and all types can be considered as cultivars. The antifungal activity of essential oil of three ginger cultivar rhizomes on Trichophyton ajelloi showed the Minimum Inhibitory Concentration (MIC) values of each ginger (1) Jahe Gajah : 0.3 l/ml, (2) Jahe Merah : 0.4 l/ml and (3) Jahe Emprit : 0.8 l/ml. The distinction of MIC values may be caused by the distinction of essential oil spectra profiles obtaining from three ginger cultivar rhizomes which were determined by Gas Chromatography. Keywords : Antifungal activity, essential oils, ginger cultivars, MIC, spectra profiles Introduction For centuries, indigenous plants have been used in herbal medicine for curing various diseases [1]. Recently, the acceptance of traditional medicine as an alternative form for health care and the development of microbial resistance to theavailable antibiotics has led authors to investigate the antimicrobial activity of medicinal plants [2, 3]. Zingiber is a genus of plants belonging to Zingiberacea, one of the largest and most important families. Zingiberaceae is among the plant families that are widely distributed throughout the tropics, particularly in Southeast Asia. It is an important natural resource that provides man with many useful products for food, spices, medicines, dyes, perfume and aesthetics [4]. Indonesia is a country of highplant biodiversity as a result of its geographical position in the tropics. In Indonesia, three types of Ginger (Zingiber officinale Roscoe) have been distinguished (1) Jahe gajah, jahe putih besar or jahe badak (2) Jahe merah or jahe sunti (3) jahe putih kecil or jahe emprit. Their rhizomes differ in shape, colour, aroma and chemical composition and all types can be considered as cultivars [5]. Jahe gajah rhizome is the biggest shape, its colour is white to yellow, jahe merah rhizome and peel colour are red, while jahe emprit are white to yellow, it is also the smallest 1

shape and the hottest taste among ginger. In recent years, several reports have been published concerning the composition and/or the biological properties (antimicrobial, antioxidant, anticancer and a stimulated effect on the immunesystem) of Zingiberaceae extracts [6-12]. These studies have emphasized the existence of marked chemical differences among oils extracted from different varieties. These variations are likely to influence the antifungal activity of the oil and are generally a function of three factors: genetically determined properties, age of the plant and environment. The objectives of this study were to compare the antifungal activity of the essential oils from three cultivars ginger rhizomes on Trichophyton ajelloi evaluating minimum inhibitory concentrations and the different of the extracts by Gas Chromatography, in an attempt to contribute to the use of these as alternative products for fungal control were determined. Materials and Methods 1. Plant Material : Jahe merah, jahe gajah dan jahe emprit (Zingiber officinale) fresh rhizomes were obtained from Mojokerto, East-Java, Indonesia in December 2007. 2. Extraction of Essential oils: Essential oils were extracted by hydrodistillation as it is a simpel and effective method to obtain essential oils from plant [13], and all operations were carried out at room temperature. The fresh rhizomes of ginger were washed to remove soil, peeled and sliced. Sliced rhizomes of fresh ginger (5 kg) were mixture with distilled water (10 L). The essential oils were extracted by hydrodistillation using a vertical hydrodistillation unit. A flask containing the homogenate was heated during 24 h and the vapor condensed and separated throughout an auto-oil/water separator. Each essential oil extraction was running in duplicate. To enable the extracted oil to be free from dissolved and suspended water it was treated with anhydrous sulphate sodium and vacuum concentrated to recover the volatile oil. 3. Capillary Gas Chromatography analisys : The extracted essential oils were disolve in n-hexane, in concentration of 1000 ppm. Capillary gas chromatography was carried out on an Agilent 6890 plus series. Analytical conditions were as follows: temperature program, 80-250 C, 5 C min-1, injector temperature 250C, helium carrier velocity 20 cm s-1, with a running time of 44 min, injection volume of 10 ul, split ratio 25:1. 4. Preparation of fungal organism 2

The fungal organism (Tricophyton ajelloi) used in this study were obtained from Microbiology Laboratory, Faculty of Medicine, Airlangga University, Indonesia. T. ajelloi is one of dhermatophyta fungi that caused the tinea corporis and tinea capitis desease [14]. Fungy was grown in Saboroud Dextrose agar (SDA) at room temperature for 7 days. The culture was standardised by matching to to the McFarland 0.5 turbidity standard using sterile saline to produce approximately 1.5x108 colony forming units (cfu) per mL. 5. Antifungal screening The antifungal screening of the essential oils were carried out by agar dillution method as previously reported [15]. The essential oils were extracted with Tween 80 in ratio 5:1, and then were added with non-antimocrobial solvent to help the oil dissolve in watered medium. Tween 80 is use for keeping the oil stability. The concentration of dispersing agent in medium is not more than 2%. SDA plates were impregnated with essential oils at concentration of 0.2 l/ml, 0.3 l/ml, 0.4 l/ml, 0.6 l/m, and 0,8 l/ml respectively.The plates were swabbed with broth culture of the organisms (diluted to 0.5 McFarland Standard with saline) and stored for absorption to take place. Negative controls were prepared using the same solvents employed to dissolve the oils. Nistatin (5g/mL) was used as positive reference standard to determine the sensitivity of one strain in fungal organism tested. The plates were incubated at 37C for 7 days. The antifungal activity was evaluated by visual analysing of the visible fungal colony growth at the lowest concentration as a Minimum Inhibitory Concentration (MIC). All experiments were conducted in triplicate. Result and Discussion 1. Determination of Essential oils Essential oils are product or mixtures of products which are formed in cytoplasm and are normally present in the form of tiny droplets between cells. They are volatile and aromatic. [16].The essential oils of three ginger cultivar rhizomes which were obtained have characteristics as shown in table 1:

Table 1: Characteristic of essential oils from three ginger cultivar rhizomes.

Jahe Merah Yield (based on 0,086% wet weight) Colour Odor Dark yellow aromatic

Jahe Gajah 0,01% Pale yellow aromatic

Jahe Emprit 0,048% Dark yellow aromatic

2. Identification and qualitatification of volatile compounds extracted from three ginger cultivar rhizomes A typical gas chromatogram of three ginger cultivar rhizomes is shown in Fig. 1, 2 and 3 and list of the peaks appear in table 2, 3 and 4.

Fig. 1: Capillary gas chromatogram of essential oils from jahe emprit rhizome

Table 2: List of peaks of Essential oils from jahe emprit analysed by capillary Gas Chromatography.

Table 2: Continued

Fig. 2: Capillary gas chromatogram of essential oils from jahe gajah rhizome Table 3: List of peaks of Essential oils from jahe gajah analysed by capillary Gas Chromatography.

Fig.3: Capillary gas chromatogram of essential oils from jahe merah rhizome Table 4: List of peaks of Essential oils from jahe merah analysed by capillary Gas Chromatography.

Figure 1 shows the capillary gas chromatogram of the essential oils from Jahe emprit about 52 peaks, in Figure 2 the essential oil from jahe gajah shows 13 peaks and in Figure 3 jahe merah shows 12 peaks respectively. Antifungal Activities The antifungal activities of three ginger cultivar rhizomes against T. ajelloi at all concentration shows in Table 2. The Minimum Inhibitory Concentration (MIC) values of Jahe Gajah, Jahe Merah and Jahe Emprit : 0.3 l/ml, 0.4 l/ml 0.8 l/ml respectively. Table 5. Minimum Inhibitory Concentration of three ginger cultivar rhizomes against T. ajelloi Concentration (l/ml) Jahe Gajah Jahe Merah Jahe Emprit 0.2 ------0.3 +++ ----0.4 +++ +++ --0.6 +++ +++ --0.8 +++ +++ +++

+++ = there is no visual colony growth in those concentrations Conclusions Essential oils from three ginger cultivar rhizomes were extracted by hydro distillation and their spectra profiles were determined by Gas Chromatography (GC). The essential oil from jahe gajah was obtained as a pale yellow, yield 0,01% based on wet weight . It was found 13 peaks in the gas chromatogram and has the MIC value of 0.3 l/ml on T. ajelloi. The essential oil from jahe merah was obtained as a dark yellow, yield 0,086 % based on wet weight . It was found 12 peaks in the gas chromatogram and has the MIC value of 0.4 l/ml on T. ajelloi. While the essential oil from jahe 7

emprit was obtained as a dark yellow, yield 0,046 % based on wet weight . It was found 52 peaks in the gas chromatogram and has the MIC value of 0.8 l/ml on T. ajelloi. The different characteristic of three ginger cultivars can be caused by three factor: genetically determined properties, age of the plant and environment. The bioassay guided fractionation procedure showed that the plant essential oil was rich in terpenes (monoterpenes, oxygenated monoterpenes and sesquiterpenes). At present, however, the mode of action of terpenic constituents on microorganisms is not fully understood. Nevertheless, in view of their hydrophobicity, it is generally considered that they are involved in such mechanism as cytoplasmic membrane, coagulation of cell contents and disruption of the proton motive force [17]. The MIC values indicate that jahe gajah was more efficient than the others and effective to be developed as an antifungal agent. However, the essential oil should be further studied in analysing the active compound and animal models for in vitro afficacy and toxicity.

References 1. Cowan, M.M. Plant products as antimicrobial agents. Clin. Microbiol. Rev. 1999, 564582. 2. Kumarasamy, Y.; Cox, P.; Jaspars, M.; Nahar, L.; Sarker, S. Screening seeds of Scottish plants for antibacterial activity. J. Ethnopharmacol. 2002, 83, 7377. 3. Srinivasan, D.; Nathan, S.; Suresh, T.; Perumalsamy, L. Antimicrobial activity of certain Indian medicinal plants used in folkloric medicine. J. Ethnopharmacol. 2001, 74, 217220. 4. Burkill, I.H. A Dictionary of the Economic Products of the Malay Peninsula. Vol I: A-H, Vol II: I-Z; Art Printing Works: Kaula Lumpur, 1966; p. 2402. 5. Prosea 13.Spices, (1999), de Guzman and J.S. Siemonsma (Editors). Backhuys Publisher, Leiden. 1999/Prosea, Bogor. p. 85. 6. Leal, P.F.; Braga, M.E.M.; Sato, D.N.; Carvalho, J.E.; Marques, M.O.M.; Meireles, M.A.A. Functional properties of spice extracts obtained via supercritical fluid extraction. J. Agric. Food Chem. 2003, 51,25202525. 7. Sekiwa, Y; Kubota, K.; Kobayashi, A. Isolation of novel glucosides related to gingerdiol from ginger and their antioxidative activities. J. Agric. Food Chem. 2000, 48, 373377. 8. Nguefack, J.; Leth, V.; Amvam, P.H.; Mathur, S.B. Evaluation of five essential oil from aromatic plant of Cameroon for controlling food spoilage and mycotoxin producing fungi. Int. J. Food Microbiol. 2004, 94, 329334. 8

9. Jirovetz, L.; Buchbauer, G.; Pottachola, M.; Kalathil, N. Analysis of the essential oils of the leaves, stems, rhizomes and roots of the medicinal plant Alpinia galanga from southern India. Acta Pharma. 2003, 53, 7381. 10. Bendjeddou, D.; Lalaoui, K.; Satta, D. Immunostimulating activity of the hot water-soluble polysaccharide extracts of Anacyclus pyrethrum, Alpinia galanga and Ethnopharmacol. 2003, 88, 155160. 11. Scartezzini, P.; Speroni, E. Review on some plants of Indian traditional medicine with antioxidant activity. J. Ethnopharmacol. 2000, 71, 2343. 12. Negi, P.S.; Jayaprakasha, G.K.; Jagan, M.R.L.; Sarariah, K.K. Antibacterial activity of turmeric oil: A byproduct from curcumin manufacture. J. Agric. Food Chem. 1999, 47, 42974300. 13. Lawrence, B.M.,. Progress in essential oils. Ginger Oil. Perfum and Flav., 2000,25:55-58 14. Jawetz, E., Melnick, J.L., and Adelberg,E.A, brooks, G.F., Butel, J.S., Ornston, 1995. Medical Microbiology, 19thEdition, Pretice Hall International Inc., London, p. 309-310. 15. Rios J.L, Recio M.C, and Villar, A.,. Screening Method for Natural Product with Antimicrobial Activity : A Review of Literature, Journal of Ethnopharmacology, 1988, 23, p.127-149 16. Alhassane Toure, Zhang X., Gas Chromatographc Analysis of Volatile Components of Guinean and Chinese Ginger Oils (Zingiber officinale) Extracted by Steam Distillation, Journal of Agronomy. 2007. 6 (2): 350-355 17. Burt, S. Essential oils: Their antibacterial properties and potential applications in food: A Review. Int. J. Food Microbiol. 2004, 94, 233-253. Citrullus colocythis. J.

10

11

12