Chemical Constituents of Kino Extract from Corymbia torelliana

Seven flavanones were identified from kino exudate of Corymbia torelliana by spectroscopic and spectrometric methods including UV, 1D and 2D NMR and UPLC-HR-MS. The study identified seven molecules, namely 3,4',5,7-tetrahydroxyflavanone (1), 3',4',5,7-tetrahydroxyflavanone (2), 4',5,7-trihydroxyflavanone (3), 3,4',5-trihydroxy-7-methoxyflavanone (4), (+)-(2S)-4',5,7-trihydroxy-6-methylflavanone (5), 4',5,7-trihydroxy-6,8-dimethylflavanone (6) and 4',5-dihydroxy-7-methoxyflavanone (7) from this eucalypt species. This is the first report of these natural products from C. torelliana kino exudate.


Introduction
Corymbia torelliana belongs to the Myrtaceae, a family of plants with at least 133 genera and more than 3800 species [1]. This family includes the eucalypts, Eucalyptus, Corymbia and Angophora, which are the world's most widely planted hardwood trees. C. torelliana is native to rainforest fringes in tropical Australia and it has an unusual mutualism with stingless bees. Stingless bees are strongly attracted to the resin of C. torelliana fruits and the bees subsequently help to disperse the seeds [2][3][4][5], thereby making this tree species invasive where it has been introduced for amenity plantings outside its natural range.
We have found that crude extract from the kino of C. torelliana has antimicrobial activity against both Gram-negative and Gram-positive bacteria in vitro. This finding led us to isolate and characterise the major chemical components of C. torelliana kino extract with a view to future screening of their antimicrobial activities.

Results and Discussion
The crude extract of kino displayed a HPLC-UV trace of eight major compounds with flavonoid-like spectra ( Figure 1). Seven compounds were recovered in sufficient purity and quantity by preparative HPLC for spectroscopic identification. The isolated compounds were identified via spectroscopic analyses, including 1 H-NMR, 13 C-NMR and high resolution mass spectroscopy. The NMR data and assignments for identified compounds are provided (Tables 1-3). The chemical formulas of the compounds were determined by liquid chromatography coupled to the high resolution mass spectrometry of [M-H] − ions ( Table 4). The structures of these compounds are shown ( Figure 2).

Experimental Section
All solvents used for extraction and chromatographic analysis were analytical grade. Ethyl acetate (EtOAc), acetonitrile (ACN) and deuterated solvent (DMSO-d6) were purchased from Merck Pty Ltd (Kilsyth, Australia). Water was obtained from an in-house Milli-Q Ultrapure water system.

Plant Material
Fresh kino samples were collected from C. torelliana trees on the Sunshine Coast (26°42'S, 153°02'E), Queensland, Australia. The botanical identity of the trees was verified by Dr. Tom Lewis, Queensland Department of Agriculture, Fisheries and Forestry, and voucher specimens were deposited in the University of the Sunshine Coast herbarium (USC14055, USC14056 and USC14057). Samples were collected into clean vials from naturally occurring kino exudates of the trees from late November to March (late spring through early autumn), transported to the laboratory on ice, and stored in the dark at −20 °C until tested. Collection of samples from crystallized kino was avoided because of the effect of sunlight exposure on the chemical composition of kino [25].

Extraction
The kino samples were extracted in EtOAc/water (4:3). The EtOAc extracts were stored at −20 °C until fractionation by preparative HPLC. The EtOAc dry extract (100 mg) was dissolved in ACN/water (1:1) for fractionation by preparative chromatography. Eight compounds were recovered, but one compound was found in very low quantities and its identification has not been completed.

Analytical HPLC-UV/DAD
Analytical chromatographic analyses were performed on a Perkin Elmer series 200 HPLC using Chromera software. The column was a Phenomenex Synergi Fusion 4 μm, 4.6 × 75 mm polar embedded RP column. Mobile Phase A was 95:5 water:ACN and Mobile Phase B was 10:90 water:ACN. The flow rate was 1.2 mL/min, gradient elution from mobile phase A to mobile phase B was 90/10 → 0/100, total run-time was 55 min, and detection was at 205, 260, 290 and 340 nm wavelength.

UPLC-HR-MS Analysis
Samples from three C. torelliana kino samples were reconstituted in MeOH (2 mg/mL) and injected onto an Eclipse Plus C18 UPLC column 1.8 μm, 2.1 × 100 mm. Mobile Phase A consisted of 0.1% acetic acid in water and Mobile Phase B consisted of 0.1% acetic acid in ACN. The flow rate was 0.4 mL/min. The gradient was 2.5% Mobile Phase B at 0.5 min to 20% Mobile Phase B at 15 min and 100% Mobile Phase B at 25 min, then held for 2.5 min. A photodiode array detector was coupled to the LC and set at 205, 260, 290 and 340 nm. The spray voltage was set to 3.5 kV with the source temperature at 100 °C. The ESI module was Heated ESI (HESI) connected onto a qExactive Mass spectrometer (qEMS). The qEMS resolution was set at 140,000 and separate UPLC runs were performed for positive and negative ionization modes.

Spectroscopic Analysis
NMR spectra were recorded on either a Varian 500 or 600 MHz unity INOVA spectrometer. The latter spectrometer was equipped with a triple resonance cold probe. The 1 H and 13 C-NMR chemical shifts were referenced to the solvent peaks for DMSO-d6 at δH 2.50 and δC 39.43, for CD3OD-d4 at δH 3.35 and δC 49.05 and for CDCl3-d1 at δH 7.25 and δC 77.0, respectively. Chemical shifts (δ) are given in ppm and coupling constants (J) in Hz (Tables 1-3).

Conclusions
We identified seven flavanones for the first time from kino exudate of the ecologically and ethnobotanically significant eucalypt tree, Corymbia torelliana. These compounds have a 4',5-dihydroxyflavanone core structure that is further substituted with hydroxyl, methoxy and/or methyl groups. Three of these flavanones have been reported previously from kino exudates of other Corymbia species, but we are reporting four other flavanones for the first time from eucalypt kino. The antimicrobial activity of these flavanones, and their potential role in the attractiveness of C. torelliana fruits to stingless bees, are the subjects of further study.