Available online at www.sciencedirect.com Phytochemistry Letters 1 (2008) 99–102 www.elsevier.com/locate/phytol Acylated flavonoid tetraglycoside from Planchonia careya leaves Jacqui M. McRae a,1, Qi Yang b, Russell J. Crawford a, Enzo A. Palombo a,* a Environment and Biotechnology Centre, Faculty of Life and Social Sciences, Swinburne University of Technology, Hawthorn, Victoria 3122, Australia b CSIRO Molecular and Health Technologies, Clayton, Victoria 3169, Australia Received 17 December 2007; received in revised form 9 April 2008; accepted 9 April 2008 Available online 5 May 2008 Abstract Phytochemical investigations of the aqueous extract of Planchonia careya leaves revealed two known flavonol glycosides, kaempferol 3-Ogentiobioside (1) and quercetin 3-O-glucoside (isoquercitrin) (2), and a novel acylated kaempferol tetraglycoside, kaempferol 3-O-[arhamnopyranosyl(1 ! 3)-(2-O-p-coumaroyl)]-b-glucopyranoside, 7-O-[a-rhamnopyranosyl-(1 ! 3)-(4-O-p-coumaroyl)]-a-rhamnopyranoside (3). Structural elucidation was achieved using UV, NMR, and mass spectrometry. # 2008 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved. Keywords: Acylated kaempferol tetraglycoside; Lecythidaceae; Planchonia careya 1. Introduction Planchonia careya (F. Muell) R. Knuth (Lecythidaceae) is a medium sized tropical tree found across northern Australia, which is traditionally used as a fish poison and a wound-healing remedy (Barr et al., 1988). There are an estimated 14 Planchonia species located across Australasia from the Andaman Islands to Papua New Guinea and Australia (Barrett, 2006), though there are few reports on the phytochemistry of this genus. Acylated triterpenoid saponins have been isolated from the bark of P. careya, which may explain the observed piscicidal properties (Khong and Lewis, 1977, 1979) and antibacterial triterpenoids have been isolated from the leaves of this species (McRae et al., 2008). Crublet et al. (2003) isolated three novel acylated kaempferol hexaglycosides from the leaves of P. grandis, which rank amongst the largest flavonoid derivatives described with respect to the number of conjugates (sugars and acylation moieties). Species of the Planchonia genus are morphologically similar to many Careya and Barringtonia species, yet no reports have been found of any acylated flavonoid polyglycosides in the latter two genera, suggesting that acylated flavonol polyglycosides may be effective chemosystematic indicators for the Planchonia genus. * Corresponding author. Tel.: +61 3 9214 8571; fax: +61 3 9819 0834. E-mail addresses: jacquimcrae@yahoo.com (J.M. McRae), epalombo@swin.edu.au (E.A. Palombo). 1 Tel.: +61 3 9214 5638. This paper details the isolation and structural elucidation of a novel acylated kaempferol tetraglycoside from the aqueous leaf extract of P. careya, which may serve as a chemotaxonomic marker for this species. This is also the first report of the known flavonol glycosides, kaempferol 3-O-gentiobioside and quercetin 3-O-glucoside, occurring in P. careya leaves. 2. Results and discussion The flavonoid component of the aqueous extract of P. careya leaves was concentrated on XAD-16 resin and eluted with methanol. Separation with RP-C18 media (100–200 mesh), followed by Sephadex LH-20 gel, and preparative HPLC gave 1 and 2, and separation with semi-preparative RP-C18 (15 mm) and preparative HPLC gave 3. Structural elucidation was achieved with 1D and 2D (homonuclear and heteronuclear) NMR experiments as well as mass spectroscopy. Acid hydrolysis of crude RP-C18 fractions, each containing either of 1, 2, or 3, produced free sugar moieties. The identity of the free sugars was determined by TLC of the acid hydrolysis products and comparison of the Rf values with authentic samples of glucose, galactose and rhamnose. The spectroscopic data for 1 was consistent with those published in the literature for kaempferol 3O-b-glucopyranoside-(1 ! 6)-glucopyranoside (kaempferol 3O-gentiobioside) (Iwashina et al., 2000; de Rijke et al., 2006). The identity of the comprising sugar moieties was confirmed as glucose by TLC of the hydrolysis products and comparison of the Rf values with those of authentic samples of glucose and 1874-3900/$ – see front matter # 2008 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.phytol.2008.04.003 100 J.M. McRae et al. / Phytochemistry Letters 1 (2008) 99–102 galactose. The spectral data of 2 concurred with the literature for quercetin 3-O-glucoside (isoquercitrin) (Kagan, 1968; de Rijke et al., 2006) and TLC of the acid hydrolysis product of the crude RP-C18 fraction confirmed that galactose was not present in the comprising compounds. The UV profile of 3 was lmax 230sh, 269, 317 nm, which was similar to those reported for the acylated kaempferol hexaglycosides isolated from P. grandis (Crublet et al., 2003). The HRMS m/z 1201.3376 [M+Na]+, calculated for C57H62O27Na, suggested that this compound consisted of fewer sugar moieties than the previously isolated hexaglycosides. TLC of the acid hydrolysis products of the crude RP-C18 fraction demonstrated that the sugar moieties of 3 were glucose and rhamnose, as was the case with the compounds isolated from P. grandis. The structural fragments of 3 (Fig. 1) were identified with 1H and 13C NMR as well as COSY and HSQC analysis. Coupling between the dH 8.02 and 6.91 doublets (2H each, J = 8.9 Hz) were indicative of the H-20 60 and H-30 50 protons of a para-substituted flavonoid B-ring, and the metacoupled doublets at dH 6.43 and 6.71 (1H each, J = 2.1 Hz) were assigned to the H-6 and H-8 positions of the flavonoid A-ring, respectively, giving the kaempferol aglycone. The downfield shifts of H-6 and H-8 (D +0.3 ppm) were indicative of 7-Oglycosylation of the aglycone (Markham et al., 1982; Fossen and Andersen, 2006). The aromatic signals at dH 7.47 and 7.44 (2H each, J = 8.7 Hz, H-2, 6 c and c00 , respectively) and the two overlapping signals at dH 6.79 (2  2H, J = 8.8 Hz, H-3, 5 c and c00 ) were indicative of two para-substituted benzene rings. The large coupling constants (J = 16 Hz) between the signals at dH 7.66 and 6.33 (1H each, H-7 c00 and H-8 c00 , respectively) and between dH 7.67 and 6.36 (1H each, H-7 c and H-8 c, respectively) were identified as two trans olefinic double bonds in very similar chemical environments. These structural fragments were attributable to two trans-p-coumaroyl subunits (Table 1). The 1H NMR spectra indicated the presence of four glycoside subunits, with anomeric protons signals at dH 5.76 (d, J = 8.02 Hz, H-1 g), 5.55 (brs, H-1 r), 4.92 (brs, H-1 r00 ), and 4.86 (brs, H-1 r000 ). The coupling constants between H-1 g and dH 5.12 (H-2 g) (J = 8.0 Hz) were indicative of a diaxial configuration and suggested that a b-glycopyranoside was present in the 4C1 conformer. TLC of the hydrolysis products confirmed that a glucopyranoside rather than a galactopyranoside was present. Doublets at dH 1.29 (J = 6.3 Hz, H-6 r000 ), 1.22 (J = 6.2 Hz, H-6 r), and 1.15 (J = 6.2 Hz, H-6 r00 ) were attributable to the methyl groups of three rhamnopyranosyl moieties. TLC of the hydrolysis products and comparison of the Table 1 The 1H and 13 C NMR data for 3 in methanol-d4 at 500 MHz Carbon no. dC d H (multiplicity, J constants, Hz) 1 2 3 4 5 6 7 8 9 10 20 –60 30 –50 40 122.58 159.09 135.23 179.41 163.08 100.71 163.25 95.72 158.01 107.81 132.49 116.53 162.03 – – – – – 6.43 – 6.71 – – 8.02 6.91 – Coumaroyl (at C-2 g) 1c 2–6 c 3–5 c 4c 7c 8c 9c 127.17 131.60 116.99 161.68 147.75 114.60 168.12 – 7.47 6.79 – 7.67 6.36 – Coumaroyl00 (at C-4 r00 ) 1 c00 2–6 c00 3–5 c00 4 c00 7 c00 8 c00 9 c00 127.24 131.60 116.99 161.73 147.83 114.60 168.49 – 7.44 6.79 – 7.66 6.33 – Glucose (at C-3) 1g 2g 3g 4g 5g 6g 100.24 75.45 83.53 70.53 78.91 62.56 5.76 5.12 3.79 3.48 3.37 3.60 3.78 (d, 8.0) (t, 8.1) (t, 8.9) (t, 9.3) (m) (dd, 12.1, 5.5) (m) Rhamnose (at C-3 g) 1r 2r 3r 4r 5r 6r 103.20 72.62 72.27 74.02 70.46 17.99 4.86 3.73 3.63 3.38 3.96 1.22 (br s) (m) (dd, 9.5, 3.3) (m) (dd, 9.6, 6.3) (d, 6.2) Rhamnose00 (at C-7) 1 r00 2 r00 3 r00 4 r00 5 r00 6 r00 99.79 71.59 78.24 73.61 69.65 18.09 5.55 4.18 4.16 5.28 3.87 1.15 (br s) (br s) (m) (t, 9.6) (m) (d, 6.2) 104.34 72.53 72.22 73.90 70.59 18.20 4.92 3.72 3.74 3.36 3.84 1.29 (br s) (m) (m) (m) (m) (d, 6.3) Rhamnose000 (at C-3 r00 ) 1 r000 2 r000 3 r000 4 r000 5 r000 6 r000 Fig. 1. The elucidated structure of 3. (d, 2.1) (d, 2.2) (d, 8.9) (d, 8.9) (d, 8.7) (d, 8.7) (d, 15.9) (d, 16.0) (d, 8.7) (d, 8.8) (d, 15.9) (d, 16.0) J.M. McRae et al. / Phytochemistry Letters 1 (2008) 99–102 determined Rf values with that of an authentic rhamnose sample confirmed the identity of these sugar moieties. The broad singlets observed in the 1H spectra for the H-1 and H-2 protons of each rhamnose unit were indicative of diequatorial configurations, confirming the a-configuration at the anomeric centres of the rhamnopyranosyl moieties. The full assignment of each glycosidic proton in each moiety was achieved with COSY and TOCSY experiments (Table 1). The positions of the glycosides and coumaroyl subunits in relation to each other and the kaempferol aglycone were determined using HMBC experiments. The anomeric proton of the glucose unit correlated with C-3 (dC 135.23) of the kaempferol aglycone (3JCH), while H-1 r00 showed 3JCH connectivity with C-7 (dC 163.25). Correlations were also observed between H-1 r and C3 g, and between H-1 r000 and C-3 r00, to give 3-O-rhamnosyl(1 ! 3)-glucopyranoside, 7-O-rhamnosyl-(1 ! 3)-rhamnoside. Cross peaks between H-2 g and dC 168.12 (C-9 c), and between H-4 r00 and dC 168.49 (C-9 c00 ) placed the coumaroyl groups at C-2 g and C-4 r00, respectively. The downfield shift of the protons adjacent to the acylated carbons (D +1.4 and 1.9 ppm for H-2 g and H-4 r00, respectively) further confirmed the position of the coumaroyl subunits. The structure of 3 was that of an acylated kaempferol tetraglycoside, kaempferol 3-O[a-rhamnopyranosyl(1 ! 3)-(2-O-p-coumaroyl)]-b-glucopyranoside, 7-O-[a-rhamnopyranosyl-(1 ! 3)-(4-O-p-coumaroyl)]-a-rhamnopyranoside. This appears to be the first report of the structure of 3, making it novel and so far unique to P. careya. Each of the acylated kaempferol hexaglycosides isolated from P. grandis consisted of the same structure as 3 with additional rhamnose or glucose moieties at the C-3 r000 and C-6 g positions. This similarity in the glycosylation pattern of the kaempferol aglycone between compounds of two Planchonia taxa indicates that similar derivatives may be found in related taxa and may therefore play an important role in the chemotaxonomic classification of members of this genus. Antibacterial minimum inhibitory concentration (MIC) assays were performed using the broth dilution method as described by Kalemba and Kunicka (2003). Compound 3 exhibited no antibacterial activity at the highest tested concentration (17 mg/ml) against Gram-positive or Gramnegative bacteria (Staphyloccus aureus and Escherichia coli). Cytotoxicity assays were performed using the standard MTT cell viability assay as described previously (Mosmann, 1983). Compound 3 demonstrated an IC50 of 256 mg/ml against monkey kidney epithelial (MA104) cells and an IC50 of 203 mg/ ml against human cervical carcinoma (HeLa) cells, suggesting that 3 possessed very weak antitumour activity against this cell line. Acylated kaempferol glycosides have been associated with protection from UV-B radiation and have demonstrated antioxidant activity (Skaltsa, 1994; Tang et al., 2001), although further investigation is required to determine any biological activity of 3. The isolation of this novel acylated kaempferol tetraglycoside from the leaves of P. careya, as well as similar structures from P. grandis, suggest that this type of compound may be common amongst this genus. Further phytochemical investigation of other Planchonia species may reveal more acylated 101 kaempferol polyglycosides, which are likely to prove useful in chemotaxonomic studies. The isolation of the relatively uncommon kaempferol 3-O-gentiobioside may also be of use as a chemosystematic marker. 3. Experimental 3.1. General experimental procedures NMR spectra were obtained on a Bruker DRX500 NMR spectrometer using the Topspin software package and with MeOD as the solvent. Gradient versions of 1H-1H DQFCOSY and TOCSY (200 ms mixing time), 1H-13C HSQC and HMBC experiments were carried out using the standard pulse sequences as supplied by Bruker. HPLC analysis was achieved with a Waters 600 instrument using an Alltech Alltima1 5 mm RP-C18 column (150 mm  4.6 mm) and ACN/H2O (30:70) at 1 ml/min. Peak detection was made with a Waters 996 Photodiode Array UV Detector (PDA). Positive and negative ion electrospray mass spectra were acquired with a VG Platform mass spectrometer using a cone voltage of 50 V and the source was maintained at 80 8C. The solvent system used was MeOH with a flow rate of 0.04 ml/ min. Optical rotations were measured with an Optical Activity Ltd PolAAr3005 automatic polarimeter, and melting points were determined with a micro-melting point apparatus and were uncorrected. 3.2. Plant material The collection of leaves from P. careya (F. Muell.) R. Knuth was authenticated by Dr. Andrew Ford, CSIRO Tropical Forest Research Centre, Queensland, Australia. Voucher specimens were lodged at the Australian National Herbarium with a voucher number of A.Ford4328. Leaves were stored at 10 8C before extraction. 3.3. Extraction and isolation The chopped fresh leaves of P. careya (4.74 kg) were extracted thrice with distilled water at room temperature under agitation via an orbital shaker for 24 h each. The combined aq. extract (460.1 g) was concentrated on Amberlite XAD-16 resin and the flavonoid component was eluted with MeOH. Further separation of this MeOH fraction was achieved with coarse Chromatorex1 C18 media (100– 200 mesh, Fuji Silysia Chemicals Ltd.) with a solvent gradient from 10% MeOH/H2O to 100% MeOH. The fraction containing flavonol glycosides 1 and 2 (determined by PDA profiles) was separated using SephadexTM LH-20 gel (Amersham) with 100% MeOH, and isolation and purification was achieved with preparative RP-HPLC (Alltech Alltima1 RP-C18, 5 mm, 250 mm  22 mm) in MeOH/ H2O (35:65), to give 1 (68.8 mg) and 2 (14.8 mg). A portion of the Chromatorex1 C18 fraction (86.2 mg) containing acylated flavonoid glycosides was kept for acid hydrolysis, and the remaining 2.1 g sample was separated with a semi- 102 J.M. McRae et al. / Phytochemistry Letters 1 (2008) 99–102 preparative Delta-PakTM C18 column (15 mm, 40 mm  300 mm Waters PrepPak1 cartridges) using an isocratic ACN/H2O (30:70) solvent system. The best resolved fraction on analytical HPLC was separated further with preparative RP-HPLC (250 mm  22 mm) using ACN/H2O (30:70) to give 3 (7.7 mg). 3.4. Acid hydrolysis The residual Chromatorex1 C18 fraction (86.2 mg) containing 3 was subjected to acid hydrolysis by adding 5 ml of 6% HCl and refluxing for 3 h at 100 8C (Tang et al., 2001). The resulting solution was concentrated to dryness and re-dissolved in H2O. TLC was performed with n-BuOH/Me2CO/NaH2PO4 (aq.) (8:8:1) (Brasseur and Angenot, 1988) on plastic-backed silica gelF254 plates (Sigma) and developed plates were visualized with aniline phthalate spray reagent (Malhorta and Dey, 1967). Acid hydrolysis was also performed on 19.2 mg of 1 and a crude fraction containing 2 (18.6 mg) using the same procedure. 3.5. Compound identification Kaempferol 3-O-[a-rhamnopyranosyl(1 ! 3)-(2-O-pcoumaroyl)]-b-glucopyranoside, 7-O-[a-rhamnopyranosyl(1 ! 3)-(4-O-p-coumaroyl)]-a-rhamnopyranoside (3). 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