Introduction

Heracleum siamicum Craib (Apiaceae) is a perennial sturdy plant known as “Ma Laep” found in the northern and northeast parts of Thailand [1]. The fruits of H. siamicum are widely used as spices. In Thai folk medicine, the fruits of H. siamicum were used as a carminative herbal drug. Because of wide usage of the fruits of H. siamicum as medicinal plant material and flavoring agent, it was decided to carry out a phytochemical study on the fruit of this plant.

Many kinds of metabolites including coumarins, furanocoumarins, anthroquinone, stilbene, furanocoumarin dimer, and flavonoids have been isolated and identified from various species of this genus [2–10]. Plant belonging to the Heracleum genus are aromatic and are excellent sources of essential oils. Essential oil composition of various members of this genus have been reported, including H. persicum [11, 12], H. candolleanum Wight et Arn. Gamble [13], H. dissectum Ledeb. [14], H. sphondylium L. subsp. ternatum Velen.) Brummitt [15], H. crenatifolium Boiss. [16, 17], H. platytaenium Boiss. [16], and H. candolleanum [18]. These oils contain monoterpene hydrocarbons (e.g. p-cymene; γ-terpene; α- and β-pinene; limonene etc.), oxygenated monoterpenes (e.g. iso-bornyl acetate, linalool, n-octanol, terpinene-1-ol-4, etc.), and sesquiterpenes (e.g. caryophyllene oxide) in their volatile fractions. Extracts from the fruits of H. persicum Desf. ex Fisher showed antibacterial activity that inhibited the growth of Staphylococcus aureus, Bacillus cereus, and Bordetella bronchiseptica [19]. Different octyl esters, especially n-octyl acetate, were reported to be the major constitute in most of the oils investigated [12, 16, 20, 21].

The present study addresses the chemical composition and antimicrobial activity of the essential oil from H. siamicum.

Results and discussion

The fruits of H. siamicum. contained 1.25% (w/w) essential oil (DW), which appeared as a liquid of light yellow color and possessed a specific sharp odor. Table 1 shows the established chemical composition. Twenty-five compounds were identified by comparison of their retention indexes and the mass spectra of each component revealed by gas chromatography (GC) to those of standards and reported data. Terpenes and their derivatives predominated, with the most abundant one being n-octyl acetate (65.30%), followed by o-cymene (10.35%), limonene (7.52%), δ-2-carene (6.87%), cis-thujone (1.92%), isobornyl acetate (0.94%), n-octanol (0.73%), 1,8-cineol (0.62%), n-tridecanol (0.44%), and safrole (0.37%). These main components (Fig. 1) comprised more than 95% of the essential oil. We should also note the presence of a total 4.31 % of alcohol hydrocarbons in the essential oil (see Table 3). Although most of these compounds are well documented as essential oil components in various plant species [22], to the best of authors’ knowledge, this is the first report of their occurrence in the essential oil of H. siamicum.

Table 1 Chemical composition of essential oil from Heracleum siamicum determined by GC-MS
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Table 2 Composition of H. siamicum essential oil by substance class
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Fig. 1
figure 1

Structure of the major components of essential oil of H. siamicum

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Table 3 Main composition of the essential oils from H. siamicum and H. crenatifolium (17)
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The dominant compound, n-octyl acetate, has been reported as a common component in most fruit oils of Heracleum genus and also reported as the constituent in Boswellia carterii Birdw.[23], Peucedanum cervaria (L.) Lapeyr. [24], and grapefruit oil [25]. Recently, the essential oil of H. sphondylium subsp. ternatum, which contains n-octanol as the major component, was reported to exhibit d a high antimicrobial against Candida albicans [15]. Limonene was shown to be biologically active as an antitumor agent [26]. The essential oil of Grammosciadium platycarpum Boiss, which contains limonene as the major constitute, exhibited a high antimicrobial activity [27].

It is interesting to note that there were significant differences between the main components of the essential oil of H. siamicum Craib and those previously determined in H. crenatifolium Boiss. [17], which belongs to the same genus. Thus, terpene alcohols such as n-octanol, limonene, and linalool are quantitatively abundant in H. candolleanum oil, while being only present in much smaller amounts in H. siamicum oil (Table 3).

The antimicrobial activity of H. siamicum essential oil was evaluated by the agar disk diffusion assay as minimum inhibition concentration (MIC) against an array of five bacteria and two fungi selected on the basis of their relevance to public health (see Table 4). The oil demonstrated strong bacteriostatic activity (with respect to Staphylococcus aureus and Bacillus subtilis), rather than fungistatic activity (which was more or less pronounced only for Candida albicans). The species of Ecsherichia coli, Pseudomonas aeruginosa and Microsporum gypseum were much less sensitive to the essential oil.

Table 4 Antimicrobial activity of essential oil from Heracleum siamicum
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Conclusions

Our GC and GC-MS study of the essential oil of H. siamicum from Northern Thailand led to the identification of 25 compounds, representing 97.69% of the total mass. The main constitutes were terpenes and their derivatives, and the most prominent one was o-cymene (10.35%). The antimicrobial activity results presented here demonstrate that this essential oil has a commercial potential.

Experimental

Plant Material Preparation and Isolation of Essential Oil

Fruits of H. siamicum Craib, were collected in January 2008 from the market of Chiangmai Province, Thailand. A voucher specimen was deposited in the Department of Pharmacognosy and Pharmaceutical Botany, Chulalongkorn University. The dried fruits were hydrodistilled in a Clevenger-type apparatus, according to the literature [28]. The oil was dried over anhydrous sodium sulfate and stored at 4°C in a vial covered with aluminum foil (to prevent the negative effect of light) until chemical analyses and microbiological tests.

Analysis of Essential Oil

Analysis was performed with a Varian Star 3400 CX gas chromatograph coupled with a Saturn III mass spectrometer (Varian Inc.) system equipped with a Varian automatic injector and a 30-m-long DB-5 MS (J&W) capillary column (0.25 mm i.d., 0.25 μm film thickness). The ionization energy was 70 eV. A sample of 1.0 μl of a 4% solution of the fruit oil in hexane was injected with a split ratio of 100:1. The temperature of the injection block was 240°C. The GC oven temperature was programmed as follows: initial temperature 60°C (1 min), followed by a temperature increase of 3°C/min up to 200°C, and the second ramp at 5°C/min to the final temperature of 220°C. The carrier gas was helium at 1.0 ml/min at constant volume. Identification of the oil components was established by comparing GC-MS spectra and RI with those of an internal Varian NIST MS 1998 library and those described by Adams [29].

Antimicrobial Activity

The microbial strains used in the antimicrobial assays were the gram-positive bacteria Bacillus subtilis (ATCC 6633), Staphylococcus aureus (ATCC 29213), and Streptococcus faecalis (ATCC 29212), the gram-negative bacteria Esherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), and the pathogenic fungi Candida albicans (ATCC 10231) and Microsporum gypseum (a clinical isolate). Antimicrobial activities of the volatile oil of H. siamicum were determined using the agar – disk diffusion method [30–33], as described below. Each bacterial strain was grown on Trypticase Soy Agar plates at 37°C for 24 h. Portions of four discrete colonies were inoculated into 5 ml of Trypticase Soy Broth (TSB) and incubated at 37°C for 2 – 3 h. The turbidity of each culture was adjusted with sterile saline. For yeast, C. albicans was grown on Sabouraud Dextrose Agar (SDA) slant at 30°C for 24 h and some of the growth was transferred to 5 ml of sterile saline. Turbidity of the inoculum suspension was adjusted with sterile saline. Microsporum gypseum (of the mold spore) was grown on Hinton agar and SDA were inoculated with bacterial and fungal suspensions by the streaking method [43]. The wells (6 mm holes) were produced in the agar with sterile cork borer No. 3. The fruit oils were diluted with sterile 0.05% Tween 80 to the final concentration of 1:20 and 50 μl of the diluted samples were pipetted into each well. The plates were left at room temperature for 1 h and then incubated at 37°C for 24 h for bacteria and at 30°C for 96 h for fungi. All tests were carried out in duplicate. The MIC values of the oil using the dilution assays were determined as described in [32]. The results were evaluated by measuring the diameters of the zones of inhibition and clear growth (in millimeters) and the minimum inhibitory concentration (MIC) was defined as the lowest concentration of the volatile oil which prevented growth of the inoculum compared with the growth in control plates.