Isolation and characterization of homoisoflavonoids from Scilla persica HAUSSKN

Medicinal plants have many traditional claims including the treatment of ailments of infectious origin. In the evaluation of traditional claims, scientific research is extremely important. In this study, five homoisoflavonoids named 3-(4’-hydroxybenzylidene)-5,7-dihydroxy-6-methoxychroman4-one(Autumnalin),3-(4’-hydroxybenzyl)-5,7-dihydroxy-6-methoxychroman-4-one (3,9-dihydroautumnalin), 3-(3’,4’-dihydroxybenzyl)-5,8-dihydroxy-7-methoxychroman-4-one,3-(3’,4’dihydroxybenzylidene)-5,8-dihydroxy-7-methoxychroman-4-one and 3-(3’,4’-dihydroxybenzylidene)5,7-dihydroxy-6-methoxychroman-4-one, were isolated from the bulbs of the plant Scilla persica HAUSSKN. Their structures were established on the basis of extensive spectroscopic analyses such as NMR, MS, IR and UV.


INTRODUCTION
Over the years, natural medicines, especially medicinal herbs, were considered for the treatment of a variety of ailments.The ingredients contained in these plants are used in the pharmaceutical industry because of their disinfecting properties (Amin, 1991;Volak, Stodola, 1995;Yasuda et al., 2013).Scilla persica HAUSSKN is a perennial plant that belongs to the Liliaceae family.The bulb of this plant is used as a foodstuff, a traditional medicine that increases blood circulation, as an anti-inflammatory agent as well as an analgesic (Crouch, Bangani, Mulholland, 1999).This plant has a small white bracteole that includes one or two high-centered flowers (10-13 flowers), with two to four leaf sheathes.Its petals are bluish in color, standing erect with inflorescence or a cluster of flowers.The axis of their flowers is usually single and rarely dual.In Iran, the habitat of this plant exists in the West Azerbaijan, Khorram Abad, Alvand, Hamedan, Qasr Shirin, Sanandaj and the Mountains of Rzab (Mobin, 1975;Rahimi, Aghaalinejad, Arslana, 2012).
In a previous paper (Ghoran et al., 2014), we reported the isolation and structural elucidation of a new homoisoflavonoid from the CHCl3 extract of fresh bulbs of S. persica along with MTT cytotoxicity assay on AGS and WEHI-164 cancerous cell lines.As part of an ongoing study of this plant, we described the isolation and structural characterization of five other homoisoflavonoids from the CHCl3 extract from the bulbs of the plant Scilla persica HAUSSKN.Their structures were identified on the basis of extensive spectroscopic analyses such as NMR, MS, IR and UV.

General experimental procedures
An Electrothermal Melting Point Apparatus was used to determine precise melting points.UV and IR spectra were recorded by a Shimadzu UV-PC 2501 and Perkin-Elmer spectrophotometer, respectively.NMR spectra were also obtained in either CD3OD or DMSO-d6 using a Bruker AV-500 instrument with TMS as the internal standard.EIMS were also recorded with a VG-Autospec-3000 spectrometer.HREIMS were recorded with a Thermo Fisher Finnegan MAT 95 XP instrument.Column chromatography (CC) was carried out using silica gel (70-230 and 230-400 mesh; E-Merck, Darmstadt, Germany).Aluminum sheets precoated with silica gel 60 F 254 (10×10 cm, 0.2 mm thick; E-Merck) were applied for TLC to check the purity of the compounds and were observed under UV light (254 and 365 nm) followed by anisaldehyde as the spray reagent and then it was heated.

Plant material
The plant used for the present study (S.persica) was collected and identified in March 2011 from the village of Valliv (Sardasht, West Azerbaijan Province, Iran) at an altitude of 1700-1800 m.A voucher specimen ( 6334) is deposited in the Herbarium of Agricultural Research Center and Natural Resources of Sari, Iran.

RESULTS AND DISCUSSION
Table I shows the results of phytochemical screening (qualification tests) on S. persica HAUSSKN.These results indicated that this plant contains flavonoids, glycosides, reducing sugars, saponins and tannins especially catecholic tannins and alkaloids.The bulb of this plant is void of terpenoids and steroids.
The spectroscopic and other physical data of the isolated compounds 1-5 were indicated below and details of 1 H and 13 C-NMR spectroscopic data were also shown in Table II.II.
The isolated compounds 1-5 in this plant showed the presence of a 5-OH group confirmed by 1 H-NMR spectroscopy (δ OH > 11.5) in Table II.
As it can be seen in Figures 1, 4 and 5 compounds have the same mass and due to the position of CH3 and -OH substituents on ring A are different.
Compound 2: Compound 2 exhibited spectral data very similar to those reported for eucomnalin (3,9-dihydroautumnalin), a compound isolated previously from Eucomis autumnalis (Tamm, 1972) and it was recently reported from S. nervosa Silayo, Ngadjui, Abegaz, 1999).The 1 H-NMR of compound 2 indicated a sharp singlet at δ H 5.93 ppm and it was assigned to H-8 in compound 2. The 13 C-NMR data is in complete agreement with the given structure (Agrawal, 1989).The location of the only methoxy signal observed in 2 was deduced to be at C-6 on the basis of the downfield resonance position of the methoxy carbon at δ C 60.47 ppm and the presence of a C-5 OH group.
Compound 3: The B ring of 3-benzyl-4-chromanone 3 has two oxygenated functions.In fact, the base peak in the mass spectra of compound 3 was due to dihydroxytropylium fragment (m/z 123).The peak (m/z 183) in the mass spectrum of 3 also indicated that the A ring has two hydroxyl and one methoxy group.The NMR signals of the hydroxyl proton, methoxy and methine protons in the A ring at δ H 11.83, 3.82 and 6.19 ppm, respectively, revealed that one hydroxyl group located at C-5 while the methoxy group is at C-7.The chemical shifts of the A ring carbons are very similar to those of 2. Therefore the remaining hydroxyl group is linked at C-8.

CONCLUSION
Scilla persica HAUSSKN is an edible plant that is used in traditional medicine to treat eczema and to relieve constipation, but, so far, no reports have been documented with evidence of its compounds.The results of phytochemical screening of this plant showed the presence of flavonoids, glycoside, tannin and saponin.The presence of flavonoids in this plant is probably responsible for the antibacterial effects.In this study, five homoisoflavonoids were isolated from bulbs of the plant Scilla persica HAUSSKN and their structures were identified using spectroscopic analyses such as NMR, MS, IR and UV.The spectroscopic and other physical data of isolated compounds were found to be similar to those reported for autumnalin isolated from the bulbs of Eucomis autumnalis, Colchicum doerfleri and Scilla nervosa.

TABLE I -
Results of phytochemical screening tests on Scilla persica HAUSSKN