Antimicrobial Activity of the Essential Oil and Some Isolated Sulfur-Rich Compounds from Scorodophloeus zenkeri

• Abstract
• Introduction
• Materials and Methods
• Plant material, essential oil and isolated pure compounds
• Strains of microorganisms and growth conditions
• Paper disc method assays for antimicrobial activity
• Determination of the minimal inhibitory concentrations
• Determination of the ATP content in E. coli and B. subtilis
• Microatmosphere method
• Results
• Evaluation of the antimicrobial activity of the samples using the paper disc method
• Determination of the minimal inhibitory concentrations (MIC)
• Antifungal activity of the essential oil
• Effect of the sulfur compounds on the ATP content of bacteria
• Discussion
• Acknowledgements
• References

Abstract

The essential oil and several pure sulfur compounds isolated from Scorodophloeus zenkeri were tested for their antibacterial and antifungal activity using a paper disc method, the poisoned food technique, a microatmosphere method and the measurement of cellular ATP content. The essential oil completely inhibited the growth of all fungi tested including yeasts, with the exception of Aspergillus flavus, and was active against the Gram-positive bacteria studied, but not the Gram-negative organisms. 2,4,5,7-Tetrathiaoctane, 2,4,5,6,8-pentathianonane, 2,3,4,6,8-pentathianonane, 2,3,5,6,8,10-hexathiaundecane, 2,3,5-trithiahexane 5-oxide, 2,4,5,7-tetrathiaoctane 2-oxide, 2,3,5,7-tetrathiaoctane 3,3-dioxide and 2,3,5-trithiahexane 3,3-dioxide differed in their effects on the strains studied with respect to both growth and synthesis of cellular ATP. 2,3,5-Trithiahexane, 2,3,4,6-tetrathiaheptane, methyl methanethiosulfonate and bis-methyl-sulfonylmethane exhibited no antimicrobial activity.

Introduction

Natural products contribute in a great extent to the fight against pathogenic microorganisms. Many plants or parts of them are used in food as spices and are thought to provide a natural conservation by inhibiting the microbial growth or to display some therapeutic activity. Many studies on plants have been carried out in order to understand their medicinal properties in general and their antimicrobial properties in particular; recently, Chen et al. [1] isolated quinoline alkaloids with significant antiplatelet aggregation activity, and other studies dealt with the antibacterial and anti-inflammatory activity of plants [2], [3].

Numerous plants used in traditional foods or healing powders remain to be studied as to these properties; Scorodophloeus zenkeri belongs to this group. S. zenkeri Harms (Caesalpiniaceae) is a tropical tree of Central Africa. It is of restricted height and its diameter rarely exceeds 80 cm [4]. The tree has a garlic-like odor due to its sulfur-containing compounds [5]. The bark, seeds and wood of S. zenkeri are used as spices in some traditional foods such as ”Nà-pôô”, ”Nkuii” and ”Bongo-tjobi” in Cameroon. In Gabon, the bark and the young leaves are used as condiments. The bark delivers the so-called ”Bubimbi-bark” drug [6]. Many healing powders in Central Africa contain parts of this plant. Recently, sulfides, alkylthiosulfides and oxygen-containing sulfur-rich compounds have been isolated from the bark essential oil and extracts of S. zenkeri [7], [8]. The antimicrobial activity of the essential oil and the main isolated sulfur compounds was investigated in the present studies.

Materials and Methods

Plant material, essential oil and isolated pure compounds

The bark was purchased from the central local market in Yaounde, Cameroon, in March 1999. It was kept at -20 °C until the experiments were carried out. A voucher specimen (number 1999/1) is retained in the collection of the ”Fachrichtung Pharmakognosie und Analytische Phytochemie”, Universität des Saarlandes, D66041 Saarbrücken, Germany. The essential oil (EO) and the pure compounds were obtained and characterized as described previously [7], [8]; the EO had a density of 1.3 and was composed of sulfides and alkylthiosulfides with 2,3,5-trithiahexane (1), 2,3,4,6-tetrathiaheptane (2), 2,4,5,7-tetrathiaoctane (3) and 2,4,5,6,8-pentathianonane (4) as the main compounds. These were isolated from both the EO and the extracts which were obtained by extraction with dichloromethane and ethyl acetate as described before [7]. In addition, 2,3,4,6,8-pentathianonane (5), 2,3,5,6,8,10-hexathiaundecane (6), 2,3,5-trithiahexane 5-oxide (7), 2,4,5,7-tetrathiaoctane 2-oxide (8), methyl methanethiosulfonate (9), bis(methylsulfonyl)methane (10), 2,3,5,7-tetrathiaoctane 3,3-dioxide (11) and 2,3,5-trithiahexane 3,3-dioxide (12) were isolated from the bark extracts [7], [8]. The molecular structures are given in Fig. [1].

Strains of microorganisms and growth conditions

Bacillus subtilis DSM10, Escherichia coli DSM498, Pseudomonas aeruginosa DSM50071 and Staphylococcus aureus DSM346 were cultured on nutrient agar (NA) or nutrient broth; Aspergillus flavus DSM62066 and Aspergillus niger DSM1988, Candida utilis DSM2361 and Saccharomyces cerevisiae DSM2155 were grown on Sabouraud agar (SA: 10 g peptone, 20 g glucose, 15 - 18 g agar-agar and 1000 ml water, pH 5.5 - 6.0). PDA (Potato Dextrose Agar, Oxoid) was used for Rhizopus nigricans CBS26328, TJA (Tomato Juice Agar, Oxoid) for Phytophtora megasperma DSM63697, and Malt Extract Peptone Agar (MEPA, 30 g Malt extract, 3 g soya peptone, 15 g agar-agar and 1000 ml water) for Trichophyton mentagrophytes DSM4870.

Paper disc method assays for antimicrobial activity

Since this method requires a relatively low amount of sample, all the probes were submitted to this assay. An overnight culture of microorganisms was adjusted to 10⁵ cells per ml, and one ml of this suspension was used as an inoculum for 50 ml of sterile agar medium (NA or SA) maintained liquid at between 46 and 50 °C. After inoculation, the medium was immediately poured into Petri dishes; after solidifying, a Whatman filter paper disc (6 mm) was soaked with 20 μl of each probe (1 mg/ml for the pure substances and 2 mg/ml for the essential oil) and laid on the inoculated media.

The petri dishes were kept for 3 - 4 hours at 0 °C to allow the prediffusion of the substances into the agar medium. The sizes of the inhibition zones (the distance between the edge of the paper disc and the growth zone of microorganisms) observed for the active samples were measured after subsequent incubation of the plates for 24 hours at 37 °C. A ten percent (v/v) solution of DMSO in water (in which the probes were dissolved) was used as a negative control. At this concentration, DMSO did not affect the growth of the microorganisms studied. As positive controls, we used penicillin G (Sigma) and nystatine (Merck) for their known activity against bacteria and yeasts, respectively [9], [10], at 2000 Units/ml each, corresponding to 40 Units/paper disc.

Determination of the minimal inhibitory concentrations

The assay was performed according to the poisoned food technique [11], [12]. One mg of pure compound was dissolved in one ml of ten percent DMSO (v/v) in water, and 0.5 ml was added to 4.5 ml of the appropriate agar medium to yield a concentration of 100 μg/ml. Subsequently, two fold dilutions were prepared. The highest concentration of the essential oil was 200 μg/ml. The media (NA or SA) were inoculated with the test strains (0.1 ml of cell suspension, corresponding to approximately 10⁴ cells) and poured into culture dishes with a diameter of 35 mm. The incubation conditions were as described above for the paper disc method. No effect of a ten percent aqueous solution of DMSO (v/v) was observed on the growth of the microorganisms. The positive controls were penicillin G and nystatine, and their highest concentration was 160 units per ml. The MIC value was determined as being the lowest concentration of the compound at which no growth occurred.

Determination of the ATP content in E. coli and B. subtilis

The microorganisms were grown at 28 °C in nutrient broth, harvested from exponential growth and washed with Tris-HCl buffer (20 mM, pH 7.7). The washed cells were resuspended in a Tris-HCl (20 mM, pH 7.7) containing 5 g glucose/l. To 100 μl of cell suspension, 20 μl of pure sulfur compound were added; after incubation times of 0, 5, 10 and 20 min, ATP was extracted by mixing this suspension with 50 μl of ice-cold 14 % perchloric acid. After 20 min on ice, 75 μl of KOH/KHCO₃ (1 M) were added and the mixture was centrifugated. 100 μl of supernatant were added to 400 μl Tris-HCl (20 mM, pH 7.7) containing 1 mM EDTA. The ATP concentration was measured by the firefly luminescence assay as described by Jahns [13]. The ATP concentration in the cells at time 0 was taken as 100 %, and the ATP concentrations determined at 5, 10 and 20 min were expressed as percentage compared to time 0.

Microatmosphere method

The microatmosphere method was carried out according to Benjilali et al. [14] and Naigre et al. [15] in order to evaluate the activity of the essential oil of S. zenkeri against filamentous fungi. This assay measures the activity of the volatile part of a sample in a closed microatmosphere between the agar medium and the Petri dish cover in comparison to a control culture. 20 - 25 ml of the appropriate nutrient media were poured into a 90 mm diameter Petri dish. The fungi were pregrown on solid media, and some material was inoculated in the centre of the plate using an inoculating loop. The Petri dishes were turned upside down and a sterile 6 mm Whatman filter paper was placed in the cover and soaked with variable amounts of essential oil. The plates were then sealed with parafilm in order to avoid a loss of the volatile parts of the essential oil from the atmosphere above the surface of the plates. For each fungus, Petri dishes were prepared with 0, 0.5, 1, 1.5, 2, 2.5, 5 and 10 μl, respectively, of essential oil. After 7 days of incubation at 26 - 28 °C the minimal inhibitory quantity (MIQ) for each fungus was determined. The MIQ was defined as being the lowest quantity of the essential oil at which no fungal growth was observed.

Results

Evaluation of the antimicrobial activity of the samples using the paper disc method

Six strains of microorganisms were studied in this assay: two Gram-positive (B. subtilis and S. aureus) as well as two Gram-negative bacteria (E. coli and Ps. aeruginosa) and two yeasts (C. utilis and S. cerevisiae). While 2,3,5-trithiahexane (1), 2,3,4,6-tetrathiaheptane (2), methyl methanethiosulfonate (9) and bis(methylsulfonyl)methane (10) showed no antimicrobial properties at concentrations of 1 mg/ml, the microorganisms showed different sensitivities towards the other samples.

As shown in Table [1] a and 1b, the essential oil, 2,4,5,7-tetrathiaoctane (3), 2,4,5,6,8-pentathianonane (4), 2,3,4,6,8 pentathianonane (5), 2,3,5,6,8,10-hexathiaundecane (6), 2,3,5-trithiahexane 5-oxide (7), 2,4,5,7-tetrathiaoctane 2-oxide (8), 2,3,5,7-tetrathiaoctane 3,3-dioxide (11) and 2,3,5-trithiahexane 3,3-dioxide (12) were active against B. subtilis and the two yeasts. The sizes of the inhibition zones varied from 0.25 to 16.25 mm. Compound 4 was the most efficient against C. utilis (16.25 mm), while compound 6, 2,3,5,6,8,10-hexathiaundecane, showed the greatest antimicrobial activity against B. subtilis (size of the inhibition zone, 3.38 mm) and S. cerevisiae (9.75 mm). 2,4,5,6,8-Pentathianonane resulted in an inhibition zone of 3.25 mm with S. aureus and was the most active antibacterial compound against this strain, growth of which was also inhibited by the essential oil and the compounds 6, 8, 11 and 12. 2,3,5,6,8,10-Hexathiaundecane, 2,4,5,7-tetrathiaoctane 2-oxide and 2,3,5-trithiahexane 3,3-dioxide showed actvity against E. coli, yielding inhibition zones with sizes of 0.5, 0.5 and 2 mm, respectively. Ps. aeruginosa was resistant to all the samples tested, with the exception of compound 6. Table [1] c shows the sizes of the inhibition zones of the reference substances.

Determination of the minimal inhibitory concentrations (MIC)

As shown in Table [2] a, the essential oil, 2,3,5-trithiahexane (1) and 2,4,5,7-tetrathiaoctane (3) exhibited different MIC values against E. coli, C. utilis, B. subtilis and S. aureus. Compound 1 showed no activity on the strains tested even at a concentration of 100 μg/ml. E. coli was resistant to concentrations of up to 100 μg/ml and 200 μg/ml for compound 3 and the essential oil, respectively. The essential oil and compound 3 showed MIC values of between 25 and 50 μg/ml for B. subtilis. Furthermore, compound 3 was effective against C. utilis in this assay, exhibiting a MIC of between 3.13 and 6.25 μg/ml. The essential oil completely inhibited the growth of C. utilis and S. aureus at 12.5 and 100 μg/ml, respectively. The MIC values for the antibiotics are shown in the Table [2] b.

Antifungal activity of the essential oil

The essential oil was active against four of the five fungi tested, with a MIQ value of less than 0.5 μl for A. niger, P. megasperma, R. nigricans and T. mentagrophytes. The mycelial growth of A. flavus was inhibited by the essential oil: on the first day, growth was observed only in the control. The percent inhibitions varied from 31 to 100 depending on the quantity of essential oil and the incubation time (Table [3]). The growth started slowly in the petri dishes containing 1 and 1.5 μl of essential oil on the second day, followed by those with 2 and 2.5 μl on the third day. At 5 μl, A. flavus started to grow only on the fourth day. 34 % of growth as compared to the control was observed on the seventh day of culture in presence of 5 μl of essential oil (Fig. [2]).

Effect of the sulfur compounds on the ATP content of bacteria

A rapid decrease of the ATP concentration is expected when a sample interferes with the production of energy in the cell. E. coli and B. subtilis were studied in this assay. 2,4,5,7-Tetrathiaoctane, 2,4,5,6,8-pentathianonane, 2,3,4,6,8-pentathianonane, 2,3,5,6,8,10-hexathiaundecane, and 2,4,5,7-tetrathiaoctane 2-oxide showed almost no effect on the ATP production in both strains, while 2,3,5-trithiahexane 3,3-dioxide (12) considerably decreased the ATP concentration in E. coli and in B. subtilis. ATP content in E. coli was reduced to approximately 40 % after 5 min incubation with compound 12, and only 14 % of the initial ATP content were measured after 10 and 20 min. The relative concentrations of ATP in B. subtilis in the presence of 2,3,5-trithiahexane 3,3-dioxide (12) were 6 %, 5 % and 3 % at 5, 10 and 20 min incubation, respectively.

Discussion

The studies presented here show that the essential oil of S. zenkeri shows antifungal activity since it was active against both yeasts and filamentous fungi in several antimicrobial assay systems tested. No complete inhibition was observed for A. flavus; the resistance of this fungus (known to be an important food contaminant and producer of aflatoxins) against essential oils was already observed by Mishra and Dubey [12]. The essential oil inhibited the growth of Gram-positive bacteria, but not Gram-negative organisms. Studies have been carried out in order to evaluate the antimicrobial and biological properties of 2,3,5-trithiahexane (1) and 2,4,5,7-tetrathiaoctane (3); both compounds inhibited the platelet aggregation in rabbits [16]. In addition, compound 3 showed good antibiotic activity against many microorganisms tested including B. subtilis, C. albicans and S. cerevisiae [17]. Compounds 6 and 12 had a large antimicrobial spectrum since they inhibited the growth of all the strains tested, compound 6 being more efficient than compound 12, except its activity against E. coli. These two new compounds were described for the first time from S. zenkeri Harms [7], [8]. Compound 4 and 6 were more active against the yeasts than nystatine (a polyene which action concerns the destabilization of the cell membrane in eucaryotes) at the concentrations studied. Penicillin G, a potent inhibitor of cell wall synthesis which action primarily concerns Gram-positive bacteria (Gram-negative bacteria are less susceptible to the antibiotic due to their outer membrane, and P. aeruginosa is known to be resistant against this antibiotic), was more effective against S. aureus than the essential oil and all the pure sulfur compounds studied. The molecular size and/or the number of the sulfur atoms in the compounds investigated seemed to play an important role in their antimicrobial activity. Compounds 1, 2, 9 and 10, the smallest of the sulfur compounds under investigation, showed no activity. The oxidation of the sulfides tested resulted in antimicrobial activity. A typical example is that of compound 1, which was inactive at the concentration used. The corresponding sulfoxide, 7, exhibited antimicrobial properties, but to a lesser extent than its oxidation product, sulfone (12). The positions of sulfur atoms in the structures also effected the activity; compounds 4 and 5 which are isomers exhibited different antimicrobial activities. Compounds 3, 4, 5, 6 and 8, which were active against B. subtilis, did not effect the ATP production in this strain under the assay conditions studied. The inhibitory properties of these compounds may be due to their interference with synthetic or some other vital metabolic processes, not directly acting on the synthesis of ATP. Compound 12 exhibited short time toxicity and inhibited the energy production in E. coli and B. subtilis, significantly reducing the cellular ATP content.

The results presented here demonstrate the antimicrobial activity of S. zenkeri and thereby support the use of parts of this plant both in traditional human medication and as spices in various foods in central Africa. The spices prepared from S. zenkeri may also protect foods from spoilage. Besides these applications, it may be suggested that natural products from this plant could be used in protection of plants against fungal diseases. Further studies will have to show whether the volatile compounds reach sufficient concentrations in vivo to inhibit microbial growth.

Acknowledgements

We thank the DAAD (Deutscher Akademischer Austauschdienst) for a grant to J. C. Kouokam.

References

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• 16 Lim H, Kubota K, Kobayashi A, Seki T, Ariga T. Inhibitory effect of sulfur containing compounds in Scorodocarpus borneensis Becc. on the aggregation of rabbit platelets.  Bioscience, Biotechnology and Biochemistry. 1999;  63 (2) 298-301
  CrossrefPubMedSearch in Google Scholar
• 17 Kubota K, Ohhira S, Kobayashi A. Identification and antimicrobial activity of the volatile flavor constituents from Scorodocarpus borneensis Becc.  Journal of Bioscience, Biotechnology and Biochemistry. 1994;  52 (4) 644-6
  PubMedSearch in Google Scholar

Dr. Thomas Jahns

FR 8.3, Mikrobiologie, Universität des Saarlandes

66041 Saarbrücken

Germany

Email: toja@rz.uni-sb.de

Phone: +49 681 302 2225

Fax: +49 681 302 3986

References

• 1 Chen I S, Chen H F, Cheng M J, Chang Y L, Teng C M, Tsutomu I. et al . Quinoline alkaloids and other constituents of Melicope semecarpifolia with antiplatelet aggregation activity.  Journal of Natural Products. 2001;  64 1143-7
  CrossrefPubMedSearch in Google Scholar
• 2 Vairappan C S, Suzuki M, Abe T, Masuda M. Halogenated metabolites with antibacterial activity from the Okinawan Laurencia species.  Phytochemistry. 2001;  58 517-23
  CrossrefPubMedSearch in Google Scholar
• 3 Resch M, Heilmann J, Steigel A, Bauer R. Further phenols and polyacetylenes from the rhizomes of Atractylodes lancea and their anti-inflammatory activity.  Planta Medica. 2001;  67 437-42
  Thieme ConnectPubMedSearch in Google Scholar
• 4 Aubréville A, Leroy J F. Flore du Cameroun. 9 Légumineuses-Césalpinioidées. Muséum National d’Histoire Naturelle, Laboratoire de Phanérogamie 1970: P. 83-6
  Search in Google Scholar
• 5 Amvam Zollo P H, Dupont Youngo M J, Fekam B F, Menut C, Lamaty G, Bessière J M. Etude comparée de la composition chimique des huiles essentielles extraites à partir d’un ”arbre à ail”: Scorodophloeus zenkeri Harms (Caesalpiniacee) et de l’ail: Allium sativum Linn. (liliacee) du Cameroun. Actes des 14èmes Journées Internationales Huiles Essentielles, Digne-les-Bains 1995: 614-7
  Search in Google Scholar
• 6 Hegnauer R, Hegnauer M. Chemotaxonomie der Pflanzen XIb-1 Leguminosae Teil 2. Birkhäuser Verlag Basel, Boston, Berlin; 1996: P. 152
  Search in Google Scholar
• 7 Kouokam J C, Zapp J, Becker H. Isolation of new alkylthiosulfides from the essential oil and extracts from the bark of Scorodophloeus zenkeri Harms.  Zeitschrift für Naturforschung. 2001;  56c 1003-7
  PubMedSearch in Google Scholar
• 8 Kouokam J C, Zapp J, Becker H. Oxygen-containing sulfur-rich compounds from the bark of the tropical garlic tree Scorodophloeus zenkeri Harms.  Phytochemistry. 2002;  60 403-7
  CrossrefPubMedSearch in Google Scholar
• 9 Dannhardt G, Grobe A, Gußmann S. 5-(3-Aminopropyl)-isoxazole: Synthese und antibakterielle Aktivität einiger Derivate.  Archiv der Pharmazie. 1988;  321 163-6
  PubMedSearch in Google Scholar
• 10 Ioset J R, Marston A, Gupta M P, Hostettmann K. Antifungal and larvicidal meroterpenoid naphthoquinones and a naphthoxirene from the roots of Cordia linnaei .  Phytochemistry. 1998;  47 729-34
  CrossrefPubMedSearch in Google Scholar
• 11 Grover R K, Moore J D. Toximetric studies of fungicides against the brown rot organisms, Sclerotinia fructicola and S. laxa .  Phytopathology. 1962;  52 876-80
  PubMedSearch in Google Scholar
• 12 Mishra A K, Dubey N K. Evaluation of some essential oils for their toxicity against fungi causing deterioration of stored food commodities.  Applied and Environmental Microbiology. 1994;  60 (4) 1101-5
  PubMedSearch in Google Scholar
• 13 Jahns T. Ammonium/urea-dependent generation of a proton electrochemical potential and synthesis of ATP in Bacillus pasteurii .  Journal of Bacteriology. 1996;  178 (2) 403-9
  PubMedSearch in Google Scholar
• 14 Benjilali B, Tantaoui-Elakari A, Ayadi A, Ihlal M. Method to study antimicrobial effects of essential oils: application to the antifungal activity of six Moroccan essences.  Journal of Food Protection. 1984;  47 748-52
  PubMedSearch in Google Scholar
• 15 Naigre R, Kalck P, Roques C, Roux I, Michel G. Comparison of antimicrobial properties of monoterpenes and their carbonylated products.  Planta Medica. 1996;  62 275-7
  PubMedSearch in Google Scholar
• 16 Lim H, Kubota K, Kobayashi A, Seki T, Ariga T. Inhibitory effect of sulfur containing compounds in Scorodocarpus borneensis Becc. on the aggregation of rabbit platelets.  Bioscience, Biotechnology and Biochemistry. 1999;  63 (2) 298-301
  CrossrefPubMedSearch in Google Scholar
• 17 Kubota K, Ohhira S, Kobayashi A. Identification and antimicrobial activity of the volatile flavor constituents from Scorodocarpus borneensis Becc.  Journal of Bioscience, Biotechnology and Biochemistry. 1994;  52 (4) 644-6
  PubMedSearch in Google Scholar

Dr. Thomas Jahns

FR 8.3, Mikrobiologie, Universität des Saarlandes

66041 Saarbrücken

Germany

Email: toja@rz.uni-sb.de

Phone: +49 681 302 2225

Fax: +49 681 302 3986