New Limonoids and a Dihydrobenzofuran Norlignan from the Roots of Toona sinensis

Two new limonoids, toonins A (1) and B (2), and one new dihydrobenzofuran norlignan, toonin C (3), were isolated from the roots of Toona sinensis together with the ten known compounds 4-methoxy-6-(2′,4′-dihydroxy-6′-methylphenyl)-pyran-2-one (4), bourjotinolone A (5), proceranone (6), matairesinol (7), 4-hydroxy-3-methoxybenzene-ethanol (8), syringic acid (9), isoscopoletin (10), lyoniresinol (11), aloeemodin (12), and β-sitosterol (13). Their structures were elucidated on the basis of one- and two-dimensional spectroscopic analysis. Isolation of compounds 4, 6–13 from this plant is reported here for the first time.


Introduction
Toona sinensis (syn. Cedrela sinensis A. Juss, Meliaceae) is a woody plant native to eastern and southeastern Asia that has more than 2,000 years of cultivation history in China [1]. The various parts tissues of this species are widely used in Traditional Chinese Medicine (TCM). The leaves and stems of this plant were used for the treatment of itch, dysentery, and enteritis [2]. The bark was used as an OPEN ACCESS astringent and depurative, the powdered roots were used as a corrective, and the fruits were used as an astringent and for the treatment of eye infections [3]. Previous phytochemical investigations on this species have resulted in the isolation of flavonoids, phenolics, alkaloids, terpenes, anthraquinones, and limonoids [4][5][6][7]. Besides its uses in TCM, the young leaves of T. sinensis (xiāngchūn) have been used extensively as a vegetable source in China for their special onion-like flavor and wealth of nutrients such as carotene and vitamins B and C, etc. The cultivated variety with red young leaves (Figure 1a) is considered of better flavour than those with green leaves (Figure 1b). Taihechun (a red variety originated from Taihe County, Anhui Province) is one of the most famous plants in this species, and was used for royal tributes during the Tang Dynasty [1]. The variety studied in this paper, also a red one, was collected from Tonglin County, where it is cultivated in the rocky mountainous regions by the local farmers. Interestingly, this variety very grows well, with very few pest incursions. A possible reason is that specific pest-repellent constituents may exist in the roots (Figure 1c). To our knowledge, no previous chemical studies have been published on the roots of this variety. To further search for the novel bioactive agents from Meliaceae plants, the roots of T. sinensis were investigated guided by LC-PAD-MS analytical data together with DPPH assay [8]. The purification work was mostly carried out employing two preparative HPLC separations after a silica gel column fractionation. Thirteen compounds, including limonoids, phenols, and other compounds were isolated and identified.
The DPPH assay showed that the EtOH extract of the root was more active than that of the leaves ( Table 2). The CHCl 3 fraction from the roots was also very active. Our research showed that the extract of T. sinensis had certain antioxidant capacity, which gave substantial support for its historical use as a healthy food. Our continuing chemical investigation on the free radical scavenging active fraction of the roots of T. sinensis yielded three new and ten known compounds, among which eight known phenols (3, 4, 7-12) and four Meliaceae limonoids (1, 2, 5, 6) were found.
Limonoids 1 and 6 belong to the ring A,D-seco group, and compound 2 is a ring A-seco group compound, while compound 5 without a seco-ring is a protolimonoid [26]. The ring A,D-seco and the protolimonoid groups have been isolated from the leaves, seeds, stems, or cortex of T. sinensis. The ring A,D-seco group with a functional oxygen group at position 11 (C-11) has been used as the distinguishing feature for species of Meliaceae [20]. However, besides the C-11 oxygenated ones, our research showed that C-11 non-oxygenated limonoids also exist in this plant. Compound 2 belongs to the evodulone type of limonoids, featuring a ring A lactone. These types of limonoids are examples of an A ring opened before the D ring, which are not common in limonoids from the Meliaceae family.

General
All solvents used in the extraction and isolation processes were redistilled prior to use. IR spectra (KBr) were obtained on a Perkin Elmer Spectrum 2000 instrument. All NMR spectra were recorded on a Bruker DRX-500 instrument in CDCl 3 or DMSO-d 6 . Chemical shifts are reported in ppm using TMS as internal standard. High-resolution ESI mass spectra were obtained on a JEOL-LC Mate LCMS system. LC-MS analyses were carried out using an Esquire 3000 instrument equipped with autosampler and DAD. Bruker DataAnalysis 3.1 software was used for data acquisition and processing. A Lichrocart cartridge, set 55-2, Merck 1.5024 was used as separation column, flow rate was 0.5 mL/min, time 15.0 min. Gradient elution was performed with water/0.05% formic acid (solvent A) and acetonitrile/0.05% formic acid (solvent B) at a constant flow rate of 0.5 mL/min. An increasing linear gradient (v/v) of solvent B was used [t (min), %B]: 0, 0; 9.5, 100; 12.2 100; 12.3, 0; 15.0, 0. Detection was carried out at 200 nm, with peak scanning between 195-600.2 nm. All of the analyses were carried out using a Turbo Ionspray source in the negative mode with the following settings: capillary exit, 100 V; nebulizer, curtain, collision, and drying gas (N 2 ). Full scan acquisition was performed by scanning from m/z 100 to 1,750 U in profile at a cycle time of 2 s with a step size of 0.1 U and a pause between each scan of 2 ms. MS/MS product ions were produced by collision-activated dissociation (CAD) of the selected precursor ions in the collision cell of the triple quadrupole mass spectrometer and analyzed using the second analyzer of the instrument. In all of the experiments, quadrupoles (Q1 and Q3) were operated at unit resolution. Different MS/MS experiments, neutral loss scan, product ion scan, and precursor ion scan of selected molecules were carried out to confirm the structure of the compounds previously identified by full scan mode. Analytical HPLC was performed on a Purospher STAR RP-18 column, 3 μm, 55 mm length  4 mm ID, Gradient elution was performed with water/0.01% TFA (solvent A) and acetonitrile/0.01% TFA (solvent B) at a constant flow rate of 1.5 mL/min. An increasing linear gradient (v/v) of solvent B was used [t (min), %B]: 0, 10; 7.5, 100; 10, 100; 10.5, 10; 13.0, 10. Detection was carried out at 200 and 220 nm, with peak scanning between 195-600.2 nm. Millennium software was used for data acquisition and processing. An Agilent ChemicalStation was used off-line for library building and RT-UV library search.

Conclusions
The chemical investigation on the free radical scavenging active fraction of the roots of T. sinensis yielded three new and ten known compounds, among which eight known phenols and four Meliaceae limonoids were found. The considerable number of phenols in the plants may account for its strong free radical scavenging activity. Isolation of compounds 4, 6-13 from this plant is reported here for the first time.