Novobiocin

Abstract

The volatile components of two essential oils obtained from plants indigenous to Nepal, anthopogon (Rhododendron anthopogon) and Curcuma zedoaria oils, and four oils, chamomile (Chamomilla recutita), French basil (Ocimum basilicum), cornmint (Mentha canadensis) and palmarosa (Cymbopogon martini var. martini), which are exotic but produced in Nepal, were analyzed with GC/MS and the antimicrobial activity of all the six oils were examined using Petri plate-paper disk method. The microorganisms tested were Staphylococcus aureus (IFO14462), Corynebacterium amycolatum (IFO 15207), Escherichia coli (IFO 15034), Candida albicans (IFO 1594) and Aspergillus ochraceus (IFO 31221). Anthopogon oil contained δ-cadinene (11.4%) and Curcuma zedoaria oil contained 1,8-cineole (15.8%) and β-eudesmol (10.6%) as major volatile components. All of the examined oils indicated antimicrobial activity at similar levels to that of oils with the same designation previously reported. It was revealed that oils produced in Nepal be effectively applicable to a variety of uses in terms of antimicrobial activity,

Key Word Index

Rhododendron anthogon, Ericaceae, Curcuma zedoaria, Zingiberaceae, Chamomilla recutita, Asteraceae, Ocimum basilicum, Mentha canadensis, Lamiaceae, Cymbopogon martini var. martini, Poaceae, essential oil composition, δ-cadinene, (E)-β-farnesene, bisabol oxide A, linalool, menthone, menthol, geranyl acetate, geraniol, 1,8-cineole, β-eudesmol, antimicrobial activity.

Introduction

The Himalayan Region including Nepal is rich in diverse, traditional medical knowledge systems due to cultural and environmental diversity. It is estimated that at least 70% of the medicinal plants in the region consist of wild species. Eighty percent of the population in this mountain region still have to rely on traditional medicines for health care and treatment for several diseases. In Nepal, 30 species of indigenous Rhododendron have been found and five species including R. anthopogon extend throughout Nepal. One Rhododendron species (R. arhoreum Sm.) was designated as the national flower of Nepal in 1962 popularly known as "LaIi Guras" among Nepali. The fresh flower oilali Guras is believed to have the properties of dissolving fish-bones stuck in the throat. After establishment of Herbs Production & Processing Co. Ltd. in 1981 as an undertaking of HMG/Nepal, several companies have developed essential oils and medicinal extracts from indigenous plants like R. anthopogon and exotic conventional herbal materials like cornmint (Mentha canailensis), both of which have been well-received in the neighbouring countries and European market. Among the function of essential oils, antimicrobial activity is one of the most important properties. That is why many workers have reported the antimicrobial activities of the oils. However, such studies on Nepalese oils have been rarely reported. Thus, it is essential to study the quantitative antimicrobial activity of Nepalese oils against pathogen-related microorganisms.

The aims of present study is to measure the component of six oils produced in Nepal and evaluate the antimicrobial activity of the six oils against some microbes selected from some human and plant pathogens.

Experimental

Oil isolation and analysis: All six oils and two preparations were obtained from Herbs Production and Processing Co. Ltd. (Kathmandu, Nepal). Some of the main components like geraniol, menthol and 1,8-cineole were obtained from Wako Pure Chemical Ltd. (Tokyo, Japan).

Rhododendron anthopogon D. Don. and Curcunw zedoaria Roscoe are indigenous plants of Nepal. Anthopogon oil was obtained by steam distillation of the aerial parts of R. anthopogon. Curcuma zedoaria oil is obtained from the rhizomes of C. zedoaria through steam distillation.

The other remaining four oils are exotic varieties to Nepal. Chamomile oil was obtained from the flowers with stalks of Chamomilla recutita Rausch, through steam distillation. French basil oil was obtained by steam distillation of semi-wilted flowering tops of Ocimum bnsilicum L., a popular culinary herb. Cornmint oil was obtained by steam distillation of the aerial part of Mentha canadensis L. Palmarosa oil was obtained by steam distillation from freshly cut whole flowering plants of Cyinbopogon martinii var. martinii.

The volatile components of the six oils were analyzed by GC/MS (HP 6890/HP 5973, Hewlett-Packard, PaIo Alto, CA). EI electron impact ion source, 70 eV connected to HP-Innowax (50 m × 0.32 mm, 0.5 µm film thickness) capillary column. The samples dissolved in hexane were injected in split mode using pressure-controlled helium as carrier gas at a flow rate of 1.3 mL/min. Injection and detector temperatures were maintained at 250°C and interface temperature at 240°C, and the oven temperature was programmed at 70°-170°C at 13°C/min and 170°-230°C at 5°C/min. The components were identified with the comparison of mass spectra with library spectra and polar retention indices [Davies (1)].

Cultures of test microbes: Strains of bacterium, yeast and fungus were selected on the basis of common human and plant pathogens, which were obtained from Institute for Fermentation Osaka, Japan (IFO). The selected bacterial strains were Gram-positive bacteria: Staphylococus aureus (IFO14462) and Corynebacteriumamycolatum (IFO15207), a Gram-negative bacterium: Escherichia coli (IFO15034). Both Staphylococus aureus and Escherichia coli were clinical isolates and international reference standard strains for disk-susceptibility of many antibiotics. Corynebacterium amycolatum were isolated from human skin and does not contain mycolic acid. These bacterial strains were maintained on Nutrient Agar slant (DIFCO Laboratories, Detroit, MI). A loopful amount of bacterial colonies from agar slants were inoculated into the flasks with 120 mL PY medium (10 g polypeptone, 2 g yeast extract, 1 g MgSO^sub 4^7H^sub 2^O in one liter water, pH 7). The flasks inoculated were incubated for 20-24 h at 37°C for the former two strains and 30°C for the last strain.

The selectedyeast strain was Candida albicans (IFO 1594), which causes bronchomycosis. The strain was maintained on GPY agar slant (10 g glucose, 5 g polypeptone, 3 g yeast extract, 3 g malt extract, 15 g agar in one liter water, pH 5.6). Among fungi, Aspergillus ochraceus (IFO 31221), isolated from cucumber roots and ochratoxigenic, was selected. The strain was maintained on Potato Dextrose Agar Medium (DIFCO Laboratories, Detroit, MI). Loopful amounts of yeast and fungal cells from agar slants were inoculated into the flasks with 120 mL of GPY liquid medium and the flasks were incubated at 25°C for 40-48 h. After incubation the culture broths were diluted to give an OD650 of 0.2 using 1% Tween 80 aqueous solutions for agar seeding of antimicrobial activity test.

Testing method of antimicrobial activities: The Petri plate-paper disk method was performed for the determination of antimicrobial activity of the oils. Agar media specified previously were used for the growth according to the species of microbes. A base layer of 10 mL molted agar medium was initially poured into the Petri plates (9 cm diameter). The diluted culture broths were transferred into the molten agar medium (approximately 1:10, vol/vol) at 40°-45°C and mixed well. Approximately 4 mL of the seed agar medium suspendedwith test microbes was then poured over the base layer. In some tests, larger Petri plates (15 cm diameter) were used. Paper disks at 6 mm diameter were soaked with 15 µL of test oils (designated as neat in the Table) or 15 µL of the diluted ones with ethanol at 1:1(vol/vol) ratio and 1:2 (vol/vol) ratio and placed on the seeded agar medium. Ethanol was used as the negative control (15 and 30 µL), while novobiocin (1.0 % w/v in ethanol) was used as the positive control for bacterium. All the procedures were performed under sterile conditions. Plates were incubated in an upright position at different temperatures as mentioned above. Diameters of inhibition were measured after 24 h for bacterium and 48 h for yeast/fungus. All the tests were run in duplicate and the average value was adopted.

Results and Discussion

Table I shows the content of the major volatile component (> 1%) of anthopogon oil from indigenous plants by GC/MS analysis, the main components of which were o-cadinene (11.4%), α-pinene (8.3%), β-caryophyllene (6.5%) and β-pinene (6.2%). Table II shows the results of Curcuma zedoaria oil from also indigenous plants, the main components of which were 1,8-cineole (15.8%), β-eudesmol (10.6%), p-cymene (7.0%) and elemol (6.3%). The main component of the four oils, chamomile, French basil, cornmint and palmarosa oils, are shown in Table III. The content of main components of the oils were as follows: chamomile oil: (E)-β-farnesene (36.9%), bisabolol oxide A (20.9%), α-farnesene (9.7%), bisabolol oxide B (5.4%) and germacrene D (5.1%); French basil oil: linalool (37.1%), geraniol (8.9%), T-cadinol (7.8%), eugenol (6.3%) and 1,8-cineole (5.8%); cornmint oil: menthol (53.0%), menthone (13.9%) and isomenthone (9.5%); palmarosa oil: geraniol (61.4%), geranyl acetate (22.6%) and linalool (4.9%).

Ethanol at 15 µL and 30 µL as a dilution solvent did not show any antimicrobial activity against the tested microbes showing ethanol is an adequate dilution solvent of the tested oils. As for the Petri plate-paper disk method, Morris et al. (2) reported a poor reflection of the results by the method to the minimum inhibitory concentration owing to differences of solubility and rate of diffusion (vapor effect), and they recommended a double agar layer method to minimize vapor effect. Carson and Riley (3), on the other hand, recognized reasonable agreement of the disk method with the minimum inhibitory concentration. We think the present Petri plate-paper disk method with a double agar layer adoptable for checking antimicrobial activity of Nepalese oils and the results can be compared with others by the disk method (2,3) and by an agar well dilution method (6,8,9,15,16).

Antimicrobial effect of oils against the five microbes is shown in Table IV. The values of > 90.0 mm mean no growth was observed on plates at 90 mm diameter. All the oils showed a relatively similar activity against Staphylococcus aureus, though chamomile and Curcuma zedoaria oils showed a lower inhibitory activity. Novobiosin (150 µg in 15 µL) as a positive control showed the inhibitory diameter at 28.5 mm, which was approximately three times higher activity than that of any of the oils against S. aureus. Palmarosa contained 61% geraniol as shown in Table III and geraniol showed the inhibitory diameter at 15 mm, 17% higher activity than palmarosa, indicating that geraniol as the main component of palmarosa oil was the inhibitive substance against S. aureus. Against other microbes, however, geraniol showed less inhibitory diameter than palmarosa oil (data not shown).

Against the other Gram-positive bacterium, Corynebacterium amycolatum, palmarosa and cornmint oils showed higher activity than the other oils. Chamomile oil, though, showed no activity. Novobiosin as a positive control showed the inhibitory diameter at 56 mm, almost three times activity as that of palmarosa or cornmint oil.

Against the Gram-negative bacterium, Escherichia coli, palmarosa and French basil oils showed higher antimicrobial activity than the other oils. Chamomile oil again showed no activity. Anthopogon oil showed the activity only under neat conditions and Curcuma zedoaria at more than 1:1 dilution. Novobiosin as positive control showed the inhibitory diameter at more than 90 mm, more than 5 times activity as that of palmarosa or French basil oils. Curcuma zedoaria oil contained 1,8-cineole (16%), as shown in Table II. As a positive control, 1,8-cineole (100%) showed an inhibitory diameter at 12 mm, 14% higher activity than that of C, zedoaria oil against E. coli. Against other microbes, however, 1,8-cineole showed less inhibitory diameter than against C. zedoaria oil (data not shown). Friedman et al (4) showed high antimicrobial activity of palmarosa and geraniol against E. coli. Pattnaik et al. (5) reported that palmarosa oil and other materials like mint or 1,8-cineole induced the formation of elongated filamentous forms of E. coli. In the comparison of Gram-positive and negative bacteria, higher sensitivity of positive ones with an agar well method (6) and an opposite result with a broth microdilution method (7) were reported. The comparison of the sensitivity against S. aureus and E. coli with anthopogon, cornmint and palmarosa resulted in higher sensitivity of S. aureus to the former two oils (12.5 mm vs. 7.5 mm and 13.0 vs. 11.5) and lower to palmarosa (12.8 vs. 17.5), which coincided with the data by Dorman et al (8) who tested with α-pinene, menthone and geraniol, the main component of the three oils, respectively. Wan et al (9) showed 8 mm inhibitory diameter of basil oil against both S. aureus and E. coli, though French basil resulted in higher sensitivity against E. coli (17.0 mm) than S. aureus (15.0 mm).

Against the yeast strain, Candida albicans, French basil oil showed the highest activity of 37 mm and palmarosa oil followed next. Geraniol showed 13 mm of the inhibitory diameter, less than that of palmarosa oil (18 mm), indicative of the antimicrobial effect by other minor components as geranyl acetate against C. albicans. Chamomile oil showed no activity, and the other three oils showed similar activities, which were between French basil and chamomile oils. Antimicrobial activities of palmarosa against C. albicans (18.0 mm), E. coli (17.5 mm) and S. aureus (12.8 mm) resulted in lower sensitivity against S. aureus, which was similar result to minimum inhibitory concentration by Hammer et al. (10).

The fungal strain, Aspergillus ochraceus, did not grow at 1:2 dilution and higher concentrations of palmarosa, French basil and cornmint oils, or diameters of inhibition for the three oils was larger than 90 mm. The other three oils including chamomile oil showed some antimicrobial activity against A. ochraceus. Basilico and Basilico (11) showed cornmint had higher activity than basil oil against A. ochraceus.

In terms of each tested oil, chamomile oil from Nepal showed antimicrobial activity against Aspergillus ochraceus as well as Staphylococcus aureus but no activity against Corynebacterium amycolatum, Escherichia coli and Candida albicans. Morris et al. (2) reported that the chamomile oil showed no zone of inhibition against all four microbes such as Staphylococcus aureus, Escherichia coli, Corynebacterium amycolatum and Candida albicans. The antimicrobial activity of chamomile oil was reported to derive mainly from a-bisabolol (12). Although the content of chamazulene in chamomile oil in the present study was 2.4%, chamazulene has been reported to inhibit lipid peroxidation (13) and the formation of leukotriene B4 (14). Among the tested oils, palmarosa oil showed almost the highest antimicrobial activity against the tested microorganisms except against Candida albicans. French basil oil showed relatively higher activity against all microbes except Corynebacterium amycolatum. Wan et al. (9) and Lachowicz et al. (15) reported the antimicrobial activity of basil oil against Escherichia coli, Staphylococcus aureus and some genera Candida. Application of basil oil as an insecticidal fumigant/powder (16,17) and as an alternative to conventional antimicrobial additives in foods (18) have been proposed. Koga et al. (19) reported a basil-resistant strain of Vibrio parahaemolyticus which showed a higher resistance to heat and H^sub 2^O^sub 2^ than the parent strain and conversely a heat-adapted V parahaemolyticus also showed a higher resistance to basil oil than non-adapted cells. Cornmint oil, the main component of which was menthol, also showed relatively high antimicrobial activity against all the tested organisms. Menthol diluted at 5 (w/v)% in ethanol showed a similar inhibitory diameter to that of cornmint oil against Staphyloccus aureus, Escherichia coli and Candida albicans (data not shown), which is agreement with the data of Morris et al. (2).

The present study showed the content ofvolatile component of two oils from plants indigenous to Nepal and other four oils and that oils produced in Nepal have similar antimicrobial activities and properties to those produced in other areas and can be effectively applied to a variety of uses.

Acknowledgement

MN expresses her deep appreciation to Dhurba Raj Bhattarai, general manager of Herbs Production and Processing Co. Ltd. of Nepal and her colleagues, Jawahar man Bajracharya, Giri Amatya and Usha, for their valuable technical assistance. The authors also would like to thank Japan International Cooperation Agency for giving us the opportunity to the cooperative research.

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Minoba Yonzon

Herbs Production & Processing Co. Ltd. Koteshwer, Kathmandu, Nepal

Dong Jin Lee

Dankook University, Cheonan, Rep. of Korea

Toshihiro Yokochi, Yasuhiro Kawano and Toro Nakahara*

National Institute of Advanced Industrial Science and Technology, 1-1 Higashi, Tsukuba, Ibaraki 305-8566 Japan

* Address for correspondence

Received: December 2001

Revised: June 2004

Accepted: July 2004

Copyright Allured Publishing Corporation Jan/Feb 2005
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