A New Ursane-Type Triterpene from the Fermented Shallot Allium Ascalonicum

Genus Allium is one of the largest genera of the Amaryllidaceae family. It was reported comprising of more than 800 species and widely distributed throughout temperate regions in the world including Europe, Asia, North America and Africa.1Allium species were also highly regarded worldwide for both therapeutic and culinary values including A. cepa (onion), A. sativum (garlic), A. porrum (leek), A. schoenoprasum (chive), and A. ascalonicum (shallot). They have been used for thousands of years as flavor-enhancing foods and folk medicines having hypocholesteremic, hypotensive, hypoglycemic, anti-thrombotic, antiallergic, anti-inflammatory, anti-microbial, antifungal, and anti-oxidant activity.2 Phytochemical studies suggested that Allium species are rich of sulfur-containing volatile compounds, phenolics, steroids, triterpenes, as well as saponins. The saponins from Allium species were highlighted for their cytotoxic and antimicrobial activity.3,4 However, the sulfur compounds responsible features and its distinctive smell. In addition, fermented shallot is delicious. In our research to find new antimicrobial agents from Allium species, herein, we report the isolation, structural elucidation of a new ursane-type triterpene and four known compounds from the fermented shallot A. ascalonicum. The anti-microbial activity of isolated triterpenoids and saponins were also evaluated by liquid-dilution method using resazurin as a redox indicator of microbial viability.


INTRODUCTION
Genus Allium is one of the largest genera of the Amaryllidaceae family. It was reported comprising of more than 800 species and widely distributed throughout temperate regions in the world including Europe, Asia, North America and Africa. 1 Allium species were also highly regarded worldwide for both therapeutic and culinary values including A. cepa (onion), A. sativum (garlic), A. porrum (leek), A. schoenoprasum (chive), and A. ascalonicum (shallot). They have been used for thousands of years as flavor-enhancing foods and folk medicines having hypocholesteremic, hypotensive, hypoglycemic, anti-thrombotic, antiallergic, anti-inflammatory, anti-microbial, antifungal, and anti-oxidant activity. 2 Phytochemical studies suggested that Allium species are rich of sulfur-containing volatile compounds, phenolics, steroids, triterpenes, as well as saponins. The saponins from Allium species were highlighted for their cytotoxic and antimicrobial activity. 3,4 However, the sulfur compounds responsible features and its distinctive smell. In addition, fermented shallot is delicious. In our research to find new antimicrobial agents from Allium species, herein, we report the isolation, structural elucidation of a new ursane-type triterpene and four known compounds from the fermented shallot A. ascalonicum. The anti-microbial activity of isolated triterpenoids and saponins were also evaluated by liquid-dilution method using resazurin as a redox indicator of microbial viability.
Other compounds were determined to be ursolic acid (2), 6 randiasaponin IV (3), 7 ilekudinoside W (4), 8 9 by the good agreement of their NMR spectral data with those reported in the literature.
The steroids, triterpenoids, and saponins from Allium species have been reviewed as potential antimicrobial activity. 3, 10 Therefore, compounds 1-5 were evaluated their antimicrobial activity against three pathogenic microbial strains including Gram-positive bacteria (Staphylococcus aureus), Gram-negative bacteria (Escherichia coli), and yeast (Candida albicans). As shown in the Table 2, except compound 2, the tested compounds exhibited antibacterial activity against both Gram-positive (S. aureus) and Gram-negative (E. coli) with IC 50 values in the range from 42.73±2.24 to 117.12±4.93 µM. Saponins (3)(4)(5) displayed cytotoxic to all tested microbial strains with IC 50 values in the range from 60.18 ± 4.10 to 95.71 ±3.86 µM. Among ursane-type triterpene (1)(2)(3)(4), saponins exhibited better anti-microbial activity in comparison with the aglycone triterpene. The standard drugs chloramphenicol and fluconazole were used as positive control for antibacterial and antifungal test. Once again, these results confirmed that triterpenoid, steroid, especially the saponins are anti-microbial components from A. ascalonicum. Compound 1, a new derivative of ursolic acid presented better anti-bacterial activity than ursolic acid (2).

General experimental procedures
Optical rotation was recorded on a Jasco P2000 polarimeter. NMR spectra were measured on a Bruker 500 MHz spectrometer using TMS as an internal standard. HR-ESI-MS was acquired on an Agilent 6530 Accurate Mass Q-TOF LC/MS system. Column chromatography was performed using silica gel, reverse phase C-18, and diaion HP-20 resins. Thin layer chromatography was carried out using pre-coated silica gel 60 F 254 and RP-18 F 254S plates. The plates were visualized under UV radiation (254 and 365 nm) and by spraying with aqueous H 2 SO 4 solution (5%) followed by heating with a heat gun.

Isolation and extraction
The dried powdered of fermented shallot A. ascalonicum (5.0 kg) was ultrasonically extracted with methanol for three times in room temperature (each 10 L, 30 min). After removal of the solvent, methanol extract (400 g) was suspended in 3 L of water and then successively partitioned with dichloromethane, ethyl acetate to give corresponding extracts dichloromethane (ASD, 73 g), ethyl acetate (ASE, 12 g), and water layer (ASW). The ASD and ASE extract were combined and separated on a silica gel column, eluting with gradient system of n-hexane/acetone (0-100% acetone) to give four fractions ASD1-ASD4. The ASD3 fraction was loaded on a RP-18 column and eluted with methanol/water (3/1, v/v) to give compounds 2 (61 mg).

3,24-acetonideclethric acid (1)
25922 (Gram-negative bacterium), and Staphylococcus aureus ATCC 13709(Gram-positive bacterium) were used to evaluate antimicrobial activity. Assay was performed in 96 well plates by liquid-dilution method using resazurin as a redox indicator of microbial viability. 11 In brief, each well containing 10µL of compounds and 190 µL of microorganism suspension (bacteria inoculum 5×10 5 CFU/mL and yeast inoculum 5×10 3 CFU/mL) was incubated at 37°C in 18h for yeast and 20h for bacteria. After addition of 10µL of resazurin (0.1 mg/mL), the microbial viability was assessed fluorimetrically (λ ex = 550 nm, λ em 590 nm) on a microplate reader (TecanGenios). The results were expressed in term of percent reduction of microorganism viability compared with control well and IC 50 values were determined from dose-response curve.

CONCLUSIONS
Five compounds were isolated from the bulbs of A. ascalonicum. Their chemical structures were determined to be 3,24-acetonideclethric acid (1), ursolic acid (2)