Scillapersicene: a new homoisoflavonoid with cytotoxic activity from the bulbs of Scilla persica HAUSSKN

Abstract The phytochemical investigation of Scilla persica HAUSSKN bulbs led to the isolation of a novel homoisoflavonoid that named Scillapersicene (1) and identified as 3-(3′,4′-dihydroxybenzylidene)-8-hydroxy-5,7-dimethoxychroman-4-one along with five known homoisoflavonoids 2–6, whose structures were elucidated using HRFAB-MS, 1D and 2D NMR spectroscopic data. The known compounds were identified as 3-(3′,4′-dihydroxybenzyl)-5,8-dihydroxy-7-methoxychroman-4-one (2), 3,9-dihydro-autumnalin (3), autumnalin (4), 3-(3′,4′-dihydroxybenzylidene)-5,8-dihydroxy-7-methoxychroman-4-one (5) and scillapersicone (6). All compounds obtained, expect 2 and 4, showed strong cytotoxic activity against AGS cell line. The toxicity on AGS cell line was measured by 1, 3, 5 and 6 with IC50 values of 8.4, 30.5, 10.7 and 24.2 μM, respectively. In addition, the physico-chemical properties of these natural compounds were optimised using density functional method (B3LYP) with standard 6-311+G* basis set. These natural products have low-energy gaps between the first ionisation potentials and highest occupied molecular orbital. In conclusion, the low-energy gap could cause reason for cytotoxic activity of homoisoflavonoids.


Introduction
The genus Scilla is believed to globally represent 81 taxa, and most of which are widespread in Iran. Scilla persica HAUSSKN is a perennial herb belonging to the Liliaceae family. In Iran, the habitat of this plant is West Azerbaijan (Sardasht), Khorramabad, Alvand, Hamedan, Qasr Shirin, Sanandaj and mountains Razaab (Mozaffarian 1997).
Homoisoflavonoids exclusively represent a modification of the flavonoidal skeleton. Feeding laboratory experiments show that the biosynthesis of 3-benzylchroman-4-ones is a modification of the C 6 -C 3 -C 6 chalcone-flavonoid pathway by incorporation of an additional carbon atom (Dewick 1975). These compounds were mainly discovered from plants belonging to the Liliaceae family and a few other plant species (Sievänen et al. 2010). Homoisoflavonoids have been reported to display multiple biological properties such as antioxidant activity (Siddaiah et al. 2006;Zhou et al. 2008), cytotoxic activity (Nguyen et al. 2006;Yan et al. 2012;Hafez Ghoran et al. 2014), inhibition of platelet aggregation (Kou et al. 2006), cough sedative (Ishibashi et al. 2001), hyperglycemia (Choi et al. 2004), anti-angiogenic (Shim et al. 2004), anti-fungal (Srinivas et al. 2003), anti-inflammatory, antiallergic, antihistaminic, angioprotective activities, and have been detected as potent phosphodiesterase inhibitors (Della Loggia et al. 1989;Amschler et al. 1996;Shim et al. 2004). Many natural products have traditionally played a prominent role in drug discovery and were the basis of most early medicines (Grabley & Thiericke 1999;Buss et al. 2003).
In this study, we report a phytochemical study on fresh bulbs of S. persica that resulted in isolation of new homoisoflavonoid (1), together with five known compounds (2-6, see Figure 1). Moreover, we also used the B3LYP/6-311+G* method implemented in Gaussian 98 package to determine the relative geometric structures, molecular orbital energy, and other physico-chemical properties of 1-6 (Frisch et al. 1998).
As shown in Supplementary Table S2, the results of cytotoxic evaluations in this study revealed that the compounds 1-6, expect 2 and 4, were able to affect the viability of cancerous AGS cells significantly. This anti-cancer effect at 0-200 μM concentrations was detected in a time-and dose-dependent manner. Compounds 2 and 4 could not be considered as an anti-tumour compounds as they affect normal cells like cancerous ones. And also compounds 1, 3, 5 and 6 showed cytotoxic activity with IC 50 values of 8.4, 30.5, 15.0 and 24.2 μM, respectively. Molecular structure of 1, obtained by theoretical calculations, is shown in Supplementary  Figure S2. The electronic densities in the highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) for compounds 1-6 at the B3LYP/6-311+G* level were studied. Our calculation exhibits that the energy levels of HOMO and LUMO are −5.60 and −2.02 eV for Scillapersicene (1), respectively. As shown in Supplementary Figure S2, the HOMO in Scillapersicene molecule is primarily situated on the carbonyl group atoms, oxygen and the carbon atoms of the A ring, while LUMO is located on the carbon atoms of the A, B and C rings. The HOMO-LUMO energy separation has been applied as an indicator of kinetic stability of the system. The energy gap (Δε = ε HOMO − ε LUMO ) of these natural compounds are calculated with the values of 3.58, 3.86, 4.26, 3.60, 3.26 and 4.29 eV at the B3LYP method, respectively. This small HOMO-LUMO gap reveals a low kinetic stability and high chemical reactivity; because it is energetically unfavourable to append electrons to a high-lying LUMO or to draw out electrons from a low-lying HOMO. Therefore, the low-energy gap can cause reason for cytotoxic activity of homoisoflavonoids. The MEP, plotted in Supplementary Figure  S2, show that oxygen atoms are negatively charged (red colour) while the hydrogen atoms of hydroxyl groups are positively charged (blue colour) in the molecule. In Supplementary  Table S3, the quantum molecular descriptors for 1-6 such as amount of HOMO, LUMO, the energy gaps, Fermi energy level (E F ), global softness (S), hardness (η), electrophilicity index (ω), electronegativity (χ) and the maximum amount of electronic charge (∆N max ) were determined (Hafez Ghoran et al. 2014).

Conclusion
Previous investigations have illustrated that homoisoflavonoids are efficient cytotoxic agents (Nguyen et al. 2006;Yen et al. 2010;Hafez Ghoran et al. 2014). The cellular mutability control by natural antimutagens can provide ways for preventing mutations that conceivably result in cancer as well as diseases caused by genotoxic agents. An intensive research in the area of antimutagenesis and anticarcinogenesis has led to the identification of a broad range of natural inhibitors acting through different mechanisms (Birt et al. 2001). In this study, one homoisoflavonoid, Scillapersicene along with five known homoisoflavonoids (2-6) were isolated by column chromatography method and identified from the medicinal plant S. persica. A bibliography demonstrated that Scillapersicene are also the first homoisoflavonoids that identified from plants of the Liliaceae family so far. In addition, this is the first report of HRFAB-MS, IR, UV, 1 H and 13 C-NMR assignments for compound 1. On the basis of cytotoxic assay, the natural compounds 1, 2, 3, 5 and 6 exhibited cytotoxicity towards one human cancer cell line, especially compounds 1 and 5 which showed in vitro cytotoxic activity against AGS cell line more potent than that of the normal cells. Through the calculations of the first ionisation potentials, HOMO, LUMO and energy gaps, it was found that these natural products have low-energy gaps between the first ionisation potentials and the HOMO. Accurate theoretical calculations can help identify and provide ways to obtain important chemical and physical information that cannot be easily obtained by experimental approaches.

Disclosure statement
No potential conflict of interest was reported by the authors.

Supplemental data and research materials
Supplementary material relating to this study is available online http://dx.doi.org/10.1080 /14786419.2015.1054286, alongside Tables S1-S3, Figures S1, S2 and the original spectra of compound 1.