Antimicrobial Activity of Eucosterol Oligosaccharides Isolated from Bulb of Squill ( Scilla scilloides )

Antimicrobial activity of methanol extract from bulb of Scilla scilloides as well as the purified eucosterol oligosaccharides (EOs) against bacteria, fungi and alga was evaluated in vitro using paper disc bioassay. EOs including scillascilloside E-1, E-2, E-3 and G-1 were isolated from the bulbs by methanol extraction, gel filtration on Sephadex LH-20 and preparative HPLC. The metabolites were identified by MS (HR-FAB-MS) and Hand C-NMR spectral data analyses. Methanol (MeOH) extract and purified compounds, EOs showed a selective inhibitory activity against eukaryotic cells including fungal species such as Aspergillus flavus, Candida albicans, Pyricularia oryzae and an alga such as Chlorella regulsris at the concentration of 200 μg/paper disc, but little active against bacteria. Out of four EO compounds, Scillascilloside E-3 revealed the highest activity. These results show that the MeOH extract and EOs from the medicinal plant, S. scilloides, may be applied as a natural fungicide or a food preservative for control of molds.


Introduction
Scilla is a genus of roughly 50 to 80 bulb-forming perennial herbs which consist of about 81 species.The genus has been currently classified as belonging to family Asparagaceae, subfamily Scilloideae which was formerly treated as a separate family, Hyacinthaceae [1].The plant is known to be native to woodlands, subalpine meadows, and seashores throughout Europe and Asia [2].Squill [Scilla scilloides (Lindl.)Druce, syn.Barnardia scilloides] has been used as a traditional Chinese medicinal plant showing antidote effect, blood circulatory activation, cough control and abscess reduction [3].Especially, the root extract has been known to have anti-inflammatory and antioxidative effects [4].Scillascillosides, eucosterol oligoglycosides (EOs) isolated from the squill plant, were found to exhibit cytotoxicity against several tumor cells [5].Especially, EOs isolated from S. scilloides have been proved to have inhibitory effect on 12-O-tetradecanoylphobol-13-acetate (TPA) stimulated 32 P incorporation into phospholipids of HeLa cells [6,7].Scillasaponins A, B and C as triterpenoid oligosaccharides, and lanosterol oligosaccharides were isolated from the plants of the subfamily Scilloideae including S. peruviana [8,9].Recently, anti-tumor activity of EOs isolated from S. scilloides has been also reported [10].
Pesticides may adversely affect humankind, are harmful to the environment, and make disrupt the natural equilibrium among microbial communities.Because natural antagonistic products derived from plants are commonly environmentally sound in disease control and food preservation, their use is now being recognized worldwide as an alternative for sustainable agriculture.
In the course of our screening for various bioactivities of native plant resources in Korea, we isolated four eucosterol oligosaccharides from bulb of S. scilloides.Herein we present antimicrobial activity of four eucosterol oligosaccharides including E-1, E-2, E-3 and G-1 compounds against bacteria, fungi and alga.

Antimicrobial Activity Test
The antimicrobial activity of the crude methanol extracts from the bulb of S. scilloides and the purified materials, eucosterol oligosaccharides, against bacteria, fungi and alga was evaluated by the paper disc method.All samples were dissolved in trace ethanol.Copyright © 2013 SciRes.PP

Results and Discussion
The use of and search for anticeptic drugs and preservatives derived from plants have accelerated in recent years [11][12][13].This study evaluated the antimicrobial activities of the methanol extract from the bulb of S. scilloides plant as well as the purified eucosterol oligosaccharides against bacteria, fungi and alga.The methanol extract from the bulb of S. scilloides was also highly active against fungi but little against bacteria tested (Table 1).2).

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In a previous research related to synthesis of antitumor saponins, eucosterol glycoside which is a lanosterol oligosaccharide, isolated from pineapple lily (Eucomis bicolor) demonstrated antitumor activity by causing 44% inhibition of TPA-stimulated 32 P incorporation into phospholipids of HeLa [6,13].Generally, the synthesis of structurally complex natural products provides not only stringent tests of known methods and reactions but also opportunities to devise new synthetic methods and strategies.Aoyama and his coworkers of Nagoya City University have synthesized the oligosaccharide subunit of scillascilloside E-1, focusing on the synthesis of its aglycon.Demands for plant-derived chemicals have been increasing in the broad industry fields including not only botanical extracts used in herbal supplements and pharmaceutical sources but also natural antifungal agents in environmentally friendly agriculture.
Our results indicated that the methanol extract as well as the purified eucosterol oligosaccharides from the medicinal plant, S. scilloides, can be applied as a natural fungicidal agent or a food preservative for control of molds.More studies on the mode of action, structure activity of the EO derivatives, and their activity spectra against various eukaryotic cells are needed in the future.

Conclusion
In conclusion, the present study clearly indicated that the methanol extract as well as the purified eucosterol oligosaccharides (EOs) from the medicinal plant, S. scilloides, may be used as a natural fungicidal agent or a food preservative for control of molds.

12 a
The concentrations of the purified EO compounds for microbial inhibition test were 200 µg (left)/500 µg (right) per paper disc (8 mm).b Slight activity.−: No activity.NE: Not examined.

Table 2 ,
the purified eucosterol oligosaccharides including E-1, E-2, E-3 and G-1 from the bulbs were selectively active against eukaryotic cells including three fungal species such as A. flavus EML-AF01, C. albicans KCTC 1940, P. oryzae EML-PO1 and an alga such as C. regularis EML-CR02 at the concentration of 200 µg per paper disc, producing an inhibition zone of up to 26 mm, but hardly active against bacteria including B. subtilis KCTC 1914, two strains of S. aureus KCTC 1916 and 1928, and S. mutans DSM 6178.Out of four purified EO compounds, Scillascilloside E-3 revealed the highest inhibitory activity against fungi and alga (Table