Antimicrobial activity of sesquiterpene lactones isolated from traditional medicinal plant, Costus speciosus (Koen ex.Retz.) Sm

Background Costus speciosus (Koen ex.Retz.) Sm (Costaceae) is an Indian ornamental plant which has long been used medicinally in traditional systems of medicine. The plant has been found to possess diverse pharmacological activities. Rhizomes are used to treat pneumonia, rheumatism, dropsy, urinary diseases, jaundice, skin diseases and leaves are usedto treat mental disorders. Method Antibacterial and antifungal activities were tested using Disc diffusion method and Minimum Inhibitory Concentration (MIC). Column chromatography was used to isolate compounds from hexane extract. X-ray crystallography technique and GC-MS analysis were used to identify the compounds Results Antibacterial and antifungal activities were observed in hexane, chloroform, ethyl acetate and methanol extracts. Hexane extract of C.speciosus showed good activity against tested fungi also. Two sesquiterpenoid compounds were isolated (costunolide and eremanthin) from the hexane extract. Both the compounds did not inhibit the growth of tested bacteria. But, both the compounds inhibited the tested fungi. The compound costunolide showed significant antifungal activity. The MIC values of costunolide were; 62.5 μg/ml against Trichophyton mentagrophytes, 62. μg/ml against T. simii, 31.25 μg/ml against T. rubrum 296, 62.5 μg/ml against T. rubrum 57, 125 μg/ml against Epidermophyton floccosum, 250 μg/ml against Scopulariopsis sp, 250 μg/ml against Aspergillus niger, 125 μg/ml against Curvulari lunata, 250 μg/ml against Magnaporthe grisea. Conclusion Hexane extract showed promising antibacterial and antifungal activity. The isolated compound costunolide showed good antifungal activity.


Background
Infectious diseases caused by bacteria, fungi, viruses and parasites are still a major threat to public health, despite the tremendous progress in human medicine. Their impact is particularly large in developing countries due to the relative unavailability of medicines and the emergence of widespread drug resistance [1]. Medicinal plants have been a source of bioactive compounds to treat many diseases. Traditionally used medicinal plants produce a variety of compounds with known therapeutic properties [2].
C. speciosus leaf infusion or decoction is utilized as a sudorific or in a bath for patients with high fever. Rhizome juice is given with sugar internally to treat leprosy [5]. C. speciosus rhizomes and roots are ascribed to be bitter [6], astringent [7,8], acrid, cooling, aphrodisiac [9,10], purgative and depurative [5], anthelmintic [8,9], antituberculosis [11], spermatorrhoea [12] and antioxidant [13]. Juice of the rhizome is applied on the head for cooling and relief from headache [7]. An alkaloid extracted from C. speciosus rhizomes had papaverine-like smooth muscle relaxant and antispasmodic activities [14]. C. speciosus rhizomes are given to treat pneumonia, rheumatism, dropsy, urinary diseases, jaundice and leaves are given to treat mental disorders; bruised leaves are applied topically to reduce fever; decoction of stem is used to control fever and dysentery [15].
Costunolide is a sesquiterpene compound. It has been previously isolated from Saussurea radix and the dried root of S. lappa. It is reported to possess various biological and immunological actions [17][18][19]. Some kinds of sequiterpene compounds induced apoptosis in cancer cells [20,21], and costunolide also exhibited preventive effects on intestinal carcinogenesis [22]. Sesquiterpene such as costunolide isolated from the leaves of Laurus nobilis was found to potently inhibit blood ethanol elevation in ethanol loaded rats [23,24]. Previously eremanthin was reported to be present in Pterodon pubescens, Eremanthus elaeagnus [25], and n-hexane extract of Larus nobilis leaves [26].
We have already reported the hypoglycemic effect of C. speciosus root crude extracts [27], hypolipidemic effect of costunolide [3], antidiabetic and antilipidemic effect of eremanthin [28] and antioxidant activities of costunolide and eremanthin [29]. We herein report the antimicrobial activity of the crude extracts and two compounds from the rhizome of C. speciosusagainstpathogenic bacteria and fungi.

Plant material
The rhizomes of Costus speciosus (Koen ex.Retz.) were collected from Thandarai, Kanchipuram District, Tamil Nadu, India in 2005. The plant was identified by Dr. D. Narasimman, Department of Botany, Madras Christian College, Chennai, India. The voucher specimen (ERIC-D-78) was deposited at Entomology Research Institute, Loyola College, Chennai, India.

Preparation of extracts
The collected rhizomes were shade dried at room temperature and ground in a manual mill. Two kilogram powder was extracted with 6 litre of hexane (1:3; w/v) for 48 hours. The extract was filtered through a Buchner funnel with Whatman number 1 filter paper. The filtrate was evaporated to dryness under reduced pressure using rotary evaporator at 40°C. The remains of the plant material were extracted using chloroform, ethyl acetate, methanol and water sequentially in a similar manner using cold percolation method [30]. The yields of extracts were: hexane (25 g); chloroform (22 g); ethyl acetate (28 g), methanol (21 g) and water (19 g).

Isolation and identification of the active compound
One kg of coarse powder was soaked in 3 litres of hexane for 72 hrs with intermittent shaking. The filtrate was filtered through Buchner funnel and concentrated using vacuum rotary evaporator at 40°C. 25 g of active crude hexane extract was chromatographed on a silica gel column (Merk 10-200 mesh, 750 gm 3.5 i.d × 60 cm) and successively eluted with stepwise gradient of petroleum ether and hexane solvent system (5%, 10%, 20%, 30%, 50%, 70% and 100%). 116 fractions were collected and each fraction was spotted on a precoated silica gel (Merk-60 F254, 0.25 mm thick) plate and eluted in Hexane: Ethyl acetate(3:1) and fractions with similar Rf values in TLC pattern were pooled together to get 8 fractions. Fraction 3 was rechromatographed on a silica gel column and eluted with a stepwise gradient of Hexane: Ethyl acetate (9:1) solvent system. An oil substance was obtained in subfraction 1; it was identified as eremanthin with a molecular formula C 15 H 18 O 2 , molecular weight of m/z 230.13, m.p 72-75°C and Rf value 0.35. Based on the GC-MS analysis the fraction 1 was confirmed as Eremanthin [28].
A single pure crystal was obtained in sub fraction 3 eluted with a stepwise gradient of Hexane: Ethyl acetate (8:2) solvent system. The crystal was subjected to crystallographic and spectral analysis for structural determination. The X-ray data for crystal were recorded using Bruker-AX, X-ray diffractometer at the Indian Institute of Technology, Chennai, India. The compound was identified as costunolide [3], with molecular formula C 15 H 20 O 2 , molecular weight m/z 234.33 and m.p 105-108°C. The Rf value of costunolide was 0.40.

Tested microorganisms
The following bacteria and fungi were used for the experiment.

Preparation of inoculums
The mother culture was streaked on Nutrient Agar medium to obtain isolated colonies. After incubation at 37°C 24 h, 4 or 5 pure colonies were selected with an inoculating needle and transferred to a tube of sterile Mueller-Hinton broth and vortexed thoroughly. The bacterial suspension was equal to the 0.5 McFarland standards. These cell suspensions were diluted with sterile MHB to provide final cell counts of about 10 4 CFU/ ml. The filamentous fungi were grown on Sabouraud Dextrose Agar (SDA) slants at 28°C for 10 days and the spores were collected using sterile doubled distilled water and homogenized. Yeast was grown on Sabouraud Dextrose Broth (SDB) at 28°C for 48 h.

Disc diffusion assay
Antibacterial activity was carried out for crude extracts using disc-diffusion method [30]. Petri plates were prepared with 20 ml of sterile Mueller Hinton Agar (MHA) (Hi-media, Mumbai). The test cultures were swabbed on the top of the solidified media and allowed to dry for 10 min. The tests were conducted at three different concentrations of the crude extract (5 mg, 2.5 mg and 1.25 mg per disc). The loaded discs were placed on the surface of the medium and left for 30 min at room temperature for compound diffusion. Negative control was prepared using respective solvent. Streptomycin (10 μg/disc) was used as positive control. The plates were incubated for 24 h at 37°C . Zones of inhibition were recorded in millimeters and the experiment was repeated twice.

Minimum Inhibitory Concentration (MIC)
The antifungal activity and Minimum Inhibitory Concentration (MIC) were performed using microdilution technique according to the standard reference method [30]. The extracts were dissolved in water + 2% Dimethyl Sulfoxide (DMSO). The initial concentration of the extract was 1 mg/ml. Test concentration was serially diluted two-fold in a 96 well plate. Each well was inoculated with 5 μl of suspension containing 10 4 CFU/ml of bacteria and 10 4 spore/ ml of fungi, respectively. For fungi, the plates were incubated for 24, 48 or 72 h at 27°up to 9 days for dermatophytes strains; bacteria plates were incubated for 24 h at 37°C. MIC was defined as the lowest extract concentration, showing no visible fungal growth after incubation time. 5 μl of tested broth was placed on the sterile MHA plates for bacteria and incubated at respective temperature. The MIC for bacteria was determined as the lowest concentration of the compound inhibiting the visual growth of the test cultures on the agar plate.

Antifungal activity of compounds
The antifungal activity of costunolide and eremanthin isolated from the hexane extract of the rhizome of C. speciosus was assessed using standard reference method [30]. 250 μg/ml of the compound was dissolved in water + 2% Dimethyl Sulfoxide (DMSO). It was serially diluted two-fold. Each well was inoculated with 5 μl of suspension containing 10 4 spore/ml of fungi. The antifungal agents' Ketoconazole and Fluconazole were included in the assays as positive controls.

Results
Hexane, chloroform, ethyl acetate, methanol and water extracts were screened against bacteria and fungi using disc diffusion method and microdilution technique. Hexane extract inhibited the growth of S. aureus (15 mm) at 2.5 mg/disc, S. epidermidis (15 mm) at 5 mg/ disc and B. subtilis (12 mm) at 2.5 mg/disc. Chloroform extract inhibited the growth of S. aureus (12 mm) at 2.5 mg/disc, S. epidermidis (13 mm) at 2.5 mg/disc and B. subtilis (9 mm) at 5 mg/disc. Ethyl acetate extract inhibited the growth of S. aureus (10 mm) at 5 mg/disc, S. epidermidis (9 mm) at 5 mg/disc and B. subtilis (9 mm) at 5 mg/disc. Methanol extract showed activity against S. aureus (14 mm) at 2.5 mg/disc, S. epidermidis (12 mm) at 2.5 mg/disc and B. subtilis (10 mm) at 5 mg/ disc. Among the tested extracts the hexane extract showed good activity against only Gram-positive bacteria and tested fungi (Table 1).
Among the tested extracts for antifungal activity the hexane extract significantly inhibited the growth of T. mentagrophytes at 0.125 mg/ml, E. floccosum at 0.250 mg/ml, T. rubrum at 0.125 mg/ml and M. grisea at 0.125 mg/ml. Other extracts inhibited the growth of fungi moderately. The results are summarized in Table 1.
Based on the above results the hexane extract was subjected to fractionation. Two active sesquiterpenoids, costunolide and eremanthin, were isolated from hexane extract of C. speciosus (Figure 1).

Discussion
Sesquiterpene lactones are the most distinctive secondary metabolites of the members of the Compositae (Asteraceae). However, they have been reported from several plant families, such as Acanthaceae, Amaranthaceae, Apiaceae, Magnoliaceae, Costaceae. They have a diversity of chemical structures and a wide range of biological activities, including antitumourogenic, insect antifeedant, plant growth regulating, antibacterial, antifungal and cytotoxic properties [31][32][33][34][35]. Here we report the antibacterial and antifungal activities of the extracts of C. speciosus rhizome. The hexane extract of C. speciosus showed significant antifungal activity. Saraf [36] reported that methanol and aqueous extracts of C. specious rhizome did not exhibit any antimicrobial activity against E. coli, S. aureus, K. pnuemoniae, P. aeruginosa.
In this study, water extract did not inhibit any tested bacteria; however methanol extract inhibited the growth of Gram-positive bacteria such as S. aureus. S. epidermidis and B. subtilis. Swarnkar and Katewa [37] reported that methanol and water extracts of C. speciosus rhizome inhibited the growth of S. aureus (12 mm). Our study showed that methanol extract inhibited the growth of S. aureus (12 mm), S. epidermidis (9 mm) and B. subtilis (14 mm). Chen et al. [18] reported that methanol extract of C. speciosus showed activity against E. coli, Salmonella enterica and S. aureus.
The isolated compounds, costunolide and eremanthin, significantly inhibited the tested pathogenic fungi at lowest concentrations. Ahmed and Abdelgalei [38] reported that costunolide isolated from Magnolia grandiflora bark showed antifungal activity against plant  Figure 1 Costunolide (1) and Eremanthin (2). pathogenic fungi. They found that costunolide showed higher antifungal activity than the reference control. The present study also showed costunolide inhibited the growth of tested fungi better than eremanthin. Both the compounds were isolated using low polar solvent (hexane). Barrero et al. [39] stated that low polar sesquiterpene lactones showed more potent antifungal activity. It is well known that the presence of α-methylene-γ-lactone is essential for potent antifungal activity of sesquiterpene lactones. The low polarity of these compounds matches with optimum lipophilic characteristics required for passing through the fungal cell wall [38].
Antifungal activity of steroid saponins and sapogenins from C. speciosus was analyzed previously by Singh et al. [40] on six species of plant pathogenic fungi at different concentrations. Saponin B was found to be highly effective against conidial germination of Botrytis cineria and Alternaria sp.

Conclusion
A thorough analysis of the results indicated that among the extracts of C. speciosus, only the hexane extract showed moderate activity against tested bacteria and promising activity against fungi. Two compounds, costunolide and eremanthin, were isolated from the hexane extract of C. speciosus with significant antifungal activity in vitro. This is the first report for the antifungal activity of costunolide and eremanthin against dermatophytes.