Formylated phloroglucinols from Eucalyptus loxophleba foliage

aly ma on and Research (NIPER), Sector-67, S.A.S. Nagar, Punjab-160062, India aduate Institute of Medical Education and Research (PGIMER), , Aust occ nd a and s loxo established by comprehensive spectral analysis and by comparison of their NMR data with anti-leishmanial activity against promastigotes of Leishmania donovani. © 2011 Elsevier B.V. All rights reserved. in a occur olatil a spec of bioactive natural and synthetic phloroglucinols, a system- Fitoterapia 82 (2011) 1118–1122 Contents lists available at ScienceDirect Fitoter j ourna l homepage: www.e ls activities. Although most of the terpenoids and polyphenolics found in eucalypts also occur in other plants, the formylated phloroglucinols are almost exclusively found in eucalypts [2]. Structurally, formylated phloroglucinol compounds are high- ly diverse and characterized by at least one fully substituted phenolic ring with one or two aldehyde groups. These compounds have shown a wide range of biological activities that can be grossly classified as a) ecologically significant (such as anti-feedant [3] and anti-fouling [4]) and b) therapeutically/ naturally occurring compounds (8 and 10). In the present article, we describe the isolation, structure elucidation and evaluation of anti-leishmanial activity of these compounds. 2. Experimental section 2.1. General trations [1]. Both volatile and non-v Eucalyptus have been investigated for pharmacologically significant (anti-bacterial [6], EBV inhibitory [7], HIV-RT inhibitory [8] inhibitory [5] etc.). ☆ NIPER communication No. 485. ⁎ Corresponding author. Tel.: +91 172 2292144; fa E-mail addresses: [email protected], ipsingh67@y 0367-326X/$ – see front matter © 2011 Elsevier B.V. doi:10.1016/j.fitote.2011.07.009 e constituents of trum of biological atic phytochemical investigation of Eucalyptus loxophleba resulted in the isolation of ten FPCs including two new 1. Introduction Eucalyptus and its relatives conta secondary chemicals many of which huge diversity of in large concen- Recently, we have reported large scale isolation of side- roxylonals and isolation and characterization of loxophlebal A, a dimeric phloroglucinol [9]. In our ongoing investigation Formylated phloroglucinols Loxophlebal A Grandinal Euglobals Antileishmanial those of related compounds in the literature. All the isolated compounds were evaluated forKeywords: Eucalyptus loxophleba Formylated phloroglucinols from Euc Jasmeen Sidana a, Sukhvinder Singh b, Sunil Ku a Department of Natural Products, National Institute of Pharmaceutical Educati b Molecular Immunology Laboratory, Department of Immunopathology, Post Gr Chandigarh 160012, India c Research School of Biology, The Australian National University, Canberra 0200 a r t i c l e i n f o a b s t r a c t Article history: Received 7 March 2011 Accepted in revised form 13 July 2011 Available online 23 July 2011 Two new naturally loxophlebal B (10) a phloroglucinols (1–7 leaves of Eucalyptu [5], anti-protozoal , aldose reductase x: +91 172 2214692. ahoo.com (I.P. Singh). All rights reserved. ralia urring formylated phloroglucinol compounds (FPCs), a dimer, cyclized FPC, loxophlebene (8) together with eight other formylated 9) were isolated from the chloroform-methanol (8:2) extract of the phleba ssp. lissophloia. The structures of new compounds were ptus loxophleba foliage☆ r Arora b, William J. Foley c, Inder Pal Singh a,⁎ apia evie r.com/ locate / f i to te All the solvents used for extraction were of analytical grade. HPLC grade methanol (JT Baker), acetonitrile (Sigma Aldrich), ultra pure water (Elga®) and acetic acid were used for sample preparation and in HPLC mobile phases. All chromatographic purifications were performed with silica gel #60–120 and silica gel G whereas all TLC (silica gel) development was performed on silica gel coated (Merck Kieselgel 60 F254, 0.2 mm thickness) plates. 1119J. Sidana et al. / Fitoterapia 82 (2011) 1118–1122 Extracts were concentrated using a vacuum rotary evapo- rator (Buchi R-114, Switzerland). IR spectra were taken on an FT-IR spectrometer (Nicolet, U.S.A.). Mass spectra were recorded on a GCMS-QS (Shimadzu, Japan) or LCMS (Waters, U.S.A.). 1H-NMR spectra were recorded at 400 MHz and 13C- NMR was recorded at 100 MHz spectrometer. 2.2. HPLC analysis The HPLC analysis was carried out on a Kromasil (Phenomenex) C18 column (250×4.6 mm, 5 μ particle size) connected to a Shimadzu HPLC system (LC-10AT VP model) fitted with SIL-20AC autosampler and SPD-M10A VP photo- diode array detector. Class VP software (Shimadzu) was used both for data collection and integration. Compounds were isolated using C18 column (250×30 mm, 10 μ particle size) connected to a preparative HPLC system (Shimadzu, CBM- 20A) equipped with LC-8A binary gradient pump, SPD-20AV UV–Vis detector, FRC-10A fraction collector and a recycle valve. 2.3. Plant material The plant material was collected from a provenance trial growing at Toolbin Western Australia and managed by the Western Australian Department of Environment and Conser- vation. A voucher specimen has been deposited in the Gauba Herbarium at the Australian National University (WJF 09/03). 2.4. Extraction and isolation The shade dried leaves (10 kg) of E. loxophleba ssp. lissophloia were coarsely ground and extracted with chloro- form-methanol (8:2) in a soxhlet extractor for 48 h. The crude extract (2.7 kg) obtained after evaporation of solvent in vacuo was used to isolate 180 g of a mixture of three sideroxylonals (A, B and C). The mother liquor left after the precipitation of sideroxylonals from chloroform-methanol extract was used for further phytochemical investigation. A portion (500 g) of this mother liquor was subjected to solvent–solvent partitioning to yield chloroform (200 g) and ethyl acetate (76 g) soluble fractions. The chloroform fraction (50 g) was loaded on a VLC assembly, set by packing 600 g of silica gel G in a G-4 sintered glass funnel of 1 L capacity to obtain a column bed height of 8 cm and i.d. of 13 cm. The column was run with hexane-ethyl acetate (0–50%) and then with chloroform-methanol (0–20% MeOH) gradients to obtain fractions A to N. Fraction A (Euglobal-rich fraction, 14.0 g) was found to be rich in phloroglucinol compounds as evident by reddish-orange spot on precoated silica gel TLC when charred with 10% methanolic H2SO4. The analytical HPLC chromatogram of fraction A showed the presence of six major peaks that were fractionated into four pools by semi-preparative HPLC [Col- umn: Princeton SPHER-C18 (250×10 mm), Mobile phase: MeOH:Water:AcOH (100:5:3), Flow rate: 3.5 mL/min, Detec- tion: 275 nm]. Eachof the pooled fractions (A1–A4)was further purified by recycle prep-HPLC [Column: C18 (250×30 mm, 10 μ particle size), Mobile phase: CH3CN (100%), Flow rate: 40 mL/min, Detection: 275 nm] to afford compound 1 (12 mg), a mixture of compounds 1 and 2 (1:4) (40 mg), 3 (30 mg), 4 (35 mg), 5 (12 mg), 6 (30 mg) and 7 (30 mg). The fractions F, G and H elutedwith 30–50% ethyl acetate in hexane presented a common spot on TLC indicating a formylated phloroglucinol (reddish color on charring with 10%methanolicH2SO4). Thesewerepooled to yield light yellow crystalline mass (210 mg) that was subjected to semiprep- HPLC [Column: Princeton SPHER-C18 (250×10 mm), Mobile phase: CH3CN:Water:TFA (80:20:0.1), Flow rate: 3.5 mL/min, Detection: 275 nm] to afford 8 (25 mg). Fraction M (12.0 g), eluted with 5–20% methanol in chloroform, was found to be rich in sideroxylonals A, B, C and loxophlebal A [as evident by silica gel TLC developedwith chloroform-methanol-acetic acid (19:0.5:0.5)]. The analytical TLC showed the presence of other spots with similar UV characteristics as those of sideroxylonals. This fraction was further fractionated by vacuum liquid chromatography in a sintered funnel of 500 mL capacity over silica gel G using hexane-ethyl acetate (0 to 50% ethyl acetate) and then chloroform-methanol (0 to 5% methanol) gradient to yield 10 sub-fractions (M1–M10). Sideroxylonals B and C were the main constituents of sub-fractions M1 and M2. Sub-fractions M3 and M4 contained sideroxylonal A along with two other less polar compounds. An enriched fraction (100 mg) was prepared from sub-fractions M3 and M4 by carefully removing the compounds eluting before (sideroxylonals B and C) and after (sideroxylonal A) the target spots. This enriched fraction (dissolved in CH3CN) was purified by semi- prep HPLC [Column: Princeton SPHER-C18 (250×10 mm), Mobile phase: CH3CN:Water:AcOH (90:9:1), Flow rate: 3.5 mL/min, Detection: 282 nm] to give two equilibrating peaks (9, 12 mg) (inseparable by prep-TLC over silica gel G). Sub-fraction M5 (120 mg) was further purified by semi-prep HPLC (conditions same as above) to yield loxophlebal A (55 mg) and another equilibrating mixture 10 (12 mg). Loxophlebene (8): Light yellow crystals; Pink fluorescence under long UV (360 nm) light; CIMS: m/z 249, 195, 165; IR (KBr) νmax 3026, 2926, 1736, 1635, 1442, 1365, 1308, 1216, 1111 cm−1; 1H NMR (400 Hz, CDCl3) δ 13.39 (1H, s), 13.17 (1H, s), 10.16 (1H, s), 10.07 (1H, s) 6.56 (1H, d, J=10.0 Hz), 5.54 (1H, d, J=10.0 Hz), 1.52 (6H, s); 13C NMR (100 MHz, CDCl3) δ 191.9, 191.8, 168.9, 165.6, 163.0, 125.76, 114.5, 104.0, 103.9, 100.8, 80.5, 28.5 ppm. Loxophlebal B (10): Brown solid; IR (KBr) νmax 3026, 2926, 1736, 1600, 1365, 1216 cm−1; HRESIMS m/z 473.1805 (calculated for C25H29O9 [M+H]+, 473.1812), 1H NMR (400 MHz, CDCl3+CD3OD, 4:1) δ 0.67 (6H, d, J=6.6 Hz, H12) 0.85 (6H, overlapping doublet, H13′), 0.96 (12H, overlapping doublet, H12′ and H13), 1.78 (2H, dsept, H11′), 2.24 (2H, m, H7a), 2.25 (2H, m, H11), 2.60 (1H, m, H10a, enol form), 2.72 (2H, dd, J=16.0, 4.4 Hz, H7b), 2.75 (1H, m, H10b, enol form), 2.83 (1H, m, H10′), 2.97 (2H, d, J=6.8 Hz, H 10 in keto form), 5.48 (2H, d, J=10.9 Hz, H7′), 5.93 (1H, s, H3), 9.88 (1H, s, CHO), 10.07 (2H, s, CHO), 10.15 (1H, s, CHO); 13C NMR (100 MHz, CDCl3+CD3OD, 4:1) δ 16.5, 16.6 (C13′), 19.6, 19.7 (C7), 22.2, 22.3 (C12′), 23.2, 23.3 (C12), 23.7 (C13), 26.5, 26.8 (C11), 28.9, 29.1 (C11′), 38.2, 38.8 (C10′), 54.0, 54.1 (C10), 77.7, 77.8 (C7′), 95.1, 95.3 (C3), 103.2, 103.5, 103.8, 104.0, 104.4, 105.0, 105.3, 106.2, 106.3 (C1, C5, C1′, C3′, C5′), 165.1, 166.4, 167.8, 168.2, 168.9, 170.9, 172.3 (C2, C4, C6 keto form, C2′, C6′, C4′), 193.3, 193.6, 193.9 (C8′ and C9′), 207.7 (C9), 208.1 (C6 enol form). (The data shown here presents the assignable 1H and 13C NMR values for both keto and enol forms of loxophlebal B). 2.5. Biological activity 2.5.1. In vitro promastigote assay Anti-leishmanial activity of the compounds (1–14) was tested in vitro using Alamar blue assay against a culture of L. donovani promastigotes grown in phenol red free RPMI-1640 (Sigma, USA), supplemented with 10% FCS (Sigma, USA) at 26 °C. L. donovani (1×105 cells/mL) promastigotes from logarithmic phase culture were grown in 96 well plate for 48 h before treatment with compounds. Dilutions were prepared in DMSO and concentration (75–300 μM) of each compound was used in triplicate. The standard miltefosine were seeded to the wells of 96-well plate (1×105 cells/well) 3. Results and discussion The mother liquor obtained after the precipitation of sideroxylonals A, B and C from the chloroform-methano (8:2) extract of Eucalyptus loxophleba ssp. lissophloia foliage was used for further isolation of FPCs. Repeated chromatog- raphy and HPLC purification led to the isolation of two new naturally occurring FPCs, a dimer named loxophlebal B (10) and a cyclized FPC loxophlebene (8) together with eight known compounds viz. euglobal Ia1 (1) [12], euglobal Ia2 (2) [12], euglobal Bl-1 (3) [13], euglobal Ib (4) [13], euglobal Ic (5) [12], euglobal IIa (6) [12], robustadial B (7) [14] and grandinal (9) (Figs. 1 and 3) [15]. Loxophlebal B (10) was obtained as a brown solid from the polar fractions of chloroform-methanol (8:2) extract of E loxophleba leaves that was rich in dimeric phloroglucino compounds [sideroxylonals A–C (11–13) and loxophlebal A ated p 1120 J. Sidana et al. / Fitoterapia 82 (2011) 1118–1122 Fig. 1. Structures of formyl and were exposed to compounds (IC50 concentration and twice the concentration of IC50 were used) in triplicate. After treatment with compounds, plate was kept at 37 °C for 48 h. After this incubation, 20 μL of Alamar-Blue reagent (Invitro- gen) was added and kept for 5 h at 37 °C. Thereafter, absorbance was measured at 570 and 600 nm. The mean percentage of cytotoxicity was calculated relative to control (unexposed to compounds) [10,11]. was used at reported IC50 value. After treatment with compounds, the plate was kept at 26 °C for 48 h. After this incubation, 20 μL of Alamar-Blue reagent (Invitrogen) was added and kept for 5 h at 37 °C. Thereafter, absorbance was measured at 570 and 600 nm. The concentration of com- pounds that produced 50% reduction in growth (IC50) of promastigotes as compared to control (untreated) was determined [10,11]. 2.5.2. In vitro cytotoxicity assay In vitro cytotoxicity was determined against PBMC (peripheral blood mononuclear cells) separated from hepa- rinized blood of a normal healthy individual by Ficoll- Hypaque (Sigma, USA) density gradient centrifugation. Alamar blue was used for in vitro cytotoxicity. Briefly, the assay was performed in 96-well tissue culture plates. Cells (14) (Fig. 1)]. The analytical HPLC chromatogram of the sub- fraction containing 10 revealed the presence of loxophlebal A (14) as the major component along with two other equilibrating peaks at tR 15.2 min. (peak A) and 24.0 min. (peak B). These two equilibrating peaks were subjected to recycle HPLC, the peak A was redirected to the HPLC column and peak B was drained off. In the next detection, peak A again showed two peaks (though not in same proportion due to dilution of the sample in the chromatographic system). This suggested that peaks A and B actually represented an equilibrating pair like that of grandinal (9). The molecular formula of 10 was established as C25H29O9 by HRESIMS ([M+H]+ atm/z 473.1805 and [M+Na]+ atm/z 495.1621). The other fragment peaks in the mass spectrum confirmed it to be a dimeric formylated phloroglucinol compound. Characteristic fragment peaks at m/z 251.0923 [C13H15O5]+ (sodiated ion present at m/z 273.0742) and m/z 223.0958 [C12H15O4]+ (sodiated ion at 245.0777) repre- sented the two monomeric units of the molecule. In MS2 studies, the fragment at m/z 251 further fragmented to give another ion at m/z 195 [C9H7O5]+, characteristic for diformy- lated phloroglucinols. The second fragment at m/z 223 (indicating the des-formyl monomeric sub-unit) lost an isobutyl side chain to give a fragment ion at m/z 167. The NMR data of 10 exhibited a double set of proton and carbon resonances as is the case with grandinal (9) [15]. The hloroglucinol compounds. l . l 1H NMR spectrum of 10 (recorded in CDCl3) presented six hydroxyl protons (δ 15.51, 15.32, 14.22, 13.19, 12.7 and 7.3 ppm) of which two (δ 12.7 and 7.3) were broad singlets. On correlating the present information with the NMR data of grandinal (9), it was confirmed that \OH at δ 7.3 ppm was present at either 2′ or 6′ position. Both the 1H and 13C NMR data indicated the presence of one isopropyl and one isovaleroyl functionality in the structure. Themethyl doublets at δ 0.67 (H-12 or −13) and δ 0.98 (H-12 or −13) showed HMBC correlations to carbon resonances at 26.5 (C-11) and 54.0 (C-10) ppm. An interesting observation in the HMQC spectrum was that the carbon at 54.0 (C-10) showed correlations with three distinct signals in proton NMR, the doublet at δ 2.97 (2H, H-10, keto form) and overlapped methine carbon at 38.2 (C-10′) could be correlated with methyl signals at δH 0.85 (δC 16.5) and δH 0.96 (δC 22.2). These methyl protons are attached to a common methine at δH 1.78 (δC 28.9)ppm. These HMBC correlations were conclusive for the presence of structural moiety as shown in Fig. 2b. On the basis of MS2 studies, the 2,4-diformyl group was placed on one phloroglucinol monomeric unit of 10. The two protons (δH 2.24 and 2.72) attached to carbon at 19.6 ppm were correlated with carbon resonances between 103.5 and 104.5 ppm. The proton at δ 2.72 also correlated to a ketonic carbon at 208.0. This supported the presence of tautomerism in ring A of the tricyclic core of dimeric phloroglucinol. Fig. 3 presents the three possible isomers of 10. The relative configuration between H-7′ and H-10′ was determined by magnitude of coupling constant of H-7′ (δ 5.48, J=10.9 Hz) that indicated a trans relationship between these protons. Biogenetically, 10 is presumed to be formed in a similar fashion as other dimeric phloroglucinols like sideroxylonals and grandinal. It may be formed by Diels–Alder cyclo- addition of an O-quinone methide derived from 3-methyl- phloroisovalerophenone and a styrene derived from naturally occurring jensenone [15]. Loxophlebene (8) was isolated as yellow needles giving a Fig. 2. HMBC correlations of structural fragments of 10. (a) Isovaleryl side chain, (b) rings B and C. 1121J. Sidana et al. / Fitoterapia 82 (2011) 1118–1122 multiplets at δ 2.60 (1H, H-10a, enol form) and 2.75 ppm (1H, H-10b, enol form). All of these protons showed same HMBC correlations i.e. to methyl carbons at δ 23.2, 23.7 (C-12 and −13) and methine carbon at δ 26.5 (C-11) ppm. This was assigned to methylene at C-10 of keto and enol forms of the molecule. The selected HMBC correlations of two tautomeric fragments of 10 are shown in Fig. 2a. The proton at δH 5.48 (δC 77.7, C-7′) showed HMBC correlations with the methylene carbon at δ 19.6 (C-7, corresponding to two multiplets at δ 2.24 and 2.72 ppm, overlapped with signals of isovaleryl side chain), a methine carbon at δ 38.2 (C-10′), high-field aromatic carbons at δ 103.2, 103.5 and 103.8 ppm and aromatic carbons attached to hydroxyl groups at δ 165.9 and 167.8 ppm. Further, the Fig. 3. Tautomeric structures o pink fluorescence in long UV (366 nm) light. The mass and NMR spectral data of 8were typical for that of diformyl monomeric phloroglucinols. The two signals at δ 13.39 and 13.17 in 1H NMR spectrum could be assigned to two hydrogen bonded phenolic groups. The two formyl signals at δH 10.16 (δC 191.9) and δH 10.07 (δC 191.8) were correlated to carbons at δC 103.9 and 104.0, respectively in the HMBC spectrum. A six proton signal at δ 1.52 was assigned to two geminal methyl groups. The two olefinic protons, having a cis relationship (J=10.1 Hz) wrt each other, were observed at δ 6.56 and 5.54, respectively. The structure was confirmed to be 5,7-dihydroxy-2,2-dimethyl-2H-chromene-6,8-dicarbaldehyde. This is thefirst time that this compoundhas been isolated froma natural source. Earlier, it was obtained during our attempts at f compounds 9 and 10. the synthesis of robustadials by DDQ mediated cyclization of isopentanoyl diformyl phloroglucinol [16]. The key HMBC correlations are shown in Fig. 4. Biogenetically, 8 may be formed by polyketide pathway similar to that of other acylphloroglucinols. After nuclear prenylation of the core phloroglucinol moiety, direct cycliza- tion between the prenyl group and the ortho hydroxy functionality leads to the benzopyran ring [17]. Sideroxylonals A–C (11–13), loxophlebal A (14), mother liquor, euglobal rich fraction (fraction A, obtained after VLC of was inactive. All the isolated compounds were found to be non-cytotoxic on peripheral blood mononuclear cells (PBMCs). Acknowledgment We thank Mr John Bartle and Dr Richard Mazanec from Fig. 4. Selected HMBC correlations of 8. 1122 J. Sidana et al. / Fitoterapia 82 (2011) 1118–1122 crude mother liquor, eluted with 0–5% ethyl acetate in hexane) and the isolated compounds (1–9) were evaluated for their anti-leishmanial activity against L. donovani pro- mastigotes (D8 clone). Fraction A showed more inhibition of promastigotes (IC50 216 μg/mL) as compared to the mother liquor (IC50 266 μg/mL) establishing the anti-leishmanial activity of euglobals. The in vitro anti-leishmanial activity of compounds 1–14 is shown in Table 1. Dimeric FPCs side- roxylonals A, B and C (11–13) with four formyl groups were found to be more active than all the tested compounds. Grandinal (9), another dimeric FPC was less active and loxophlebal A (14) was inactive. Both 9 and 14 have one formyl group less than that of sideroxylonals. This suggests a plausible role of formyl functionality in the bioactivity of these compounds. All the tested euglobals were moderately active in this assay whereas the cyclized monomeric FPC (8) Table 1 In vitro anti-leishmanial activity and cytotoxicity of compounds 1–14. Compound IC50 (μM) on Leishmania donovani D8 Cytotoxicity at IC50 (μM) on PBMCs (%) Cytotoxicity at double the IC50 (μM) on PBMCs (%) 1 228.28 0 1.5 1+2 (1:4) 204.18 1.5 7 3 188.13 1.8 5.6 4 210.00 1.4 8.9 5 219.43 9.1 0.6 6 235.39 1.9 0 7 NTa – – 8 NA – – 9 244.00 1.9 4.1 10 NT – – 11 96.50 1.8 5.5 12 88.24 1.0 7.7 13 102.00 1.1 0.8 14 NA – – Miltefosine (Std.) 9.31 17 26 a Anti-leishmanial activity reported earlier, PBMC: Peripheral Blood Mononuclear Cells, NA: Not active, NT: Not tested. l the Department of the Environment, Western Australia for access to leaf material of Eucalyptus loxophleba. Authors are also thankful to the Director of NIPER for support. References [1] Keszei A, Brubaker C, Foley WJ. A molecular perspective on terpene formation in Australian Myrtaceae. Aust J Bot 2008;56:197–213. [2] Eschler BM, Pass DM,Willis R, FoleyWJ. Distribution of foliar formylated phloroglucinol derivatives amongst Eucalyptus species. Biochem Syst Ecol 2000;28:813–24. 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General 2.2. HPLC analysis 2.3. Plant material 2.4. Extraction and isolation 2.5. Biological activity 2.5.1. In vitro promastigote assay 2.5.2. In vitro cytotoxicity assay 3. Results and discussion Acknowledgment References