Oleanane Triterpenoids from the Algerian Salvia phlomoides Lamia Bennioua, Moses K. Langatb,c, Dulcie A. Mulhollandb,c , Fadila Benayache and Samir Benayachea*  a Unité de Recherche Valorisation des Ressources Naturelles, Molécules Bioactives et Analyse Physico-Chimique et Biologique (VARENBIOMOL), Université Constantine 1, 25000 Constantine, Algérie bNatural Products Research Group, Department of Chemistry, Faculty of Engineering and Physical Sciences, University of Surrey, Guildford GU2 7XH, UK cSchool of Chemistry and Physics, University of KwaZulu-Natal, Durban 4041, South Africa Corresponding Author E-mail: s.benayache@yahoo.com Keywords: Lamiaceae, Salvia phlomoides, oleanane triterpenoids, cytotoxicity screening.  ABSTRACT Three previously unreported triterpenoids 2α,3β,11α-trihydroxyolean-18-ene 1, 3β-acetoxy,2α,11α- dihydroxyolean-18-ene 2 and 2α-acetoxy,3β,11α-dihydroxyolean-18-ene 3 were isolated from the chloroform extract of aerial parts (leaves and flowers) of Salvia phlomoides collected from the Constantine region in the east of Algeria. Structures were determined using NMR spectroscopy and HR-ESI mass spectrometry. Compounds 1, 2 and 3 were tested against the NCI panel of human tumour cell lines at a single dose of 10 µM. Compound 2 was the most active showing a 38 % growth inhibition against the leukemia RPMI-8226 line. Introduction Salvia, one of the largest genera of the Lamiaceae family, is represented by over 900 species (Standley and Williams, 1973). Many species from the Salvia genus have been reported to produce compounds with pharmacological properties including antioxidant, neuroprotective (Asadi et al., 2010; Ben Farhat et al., 2009; Orhan et al., 2012), anti-inflammatory (Kamatou et al., 2010), antimicrobial (Veličković et al., 2002), antiviral (Schinitzler and Nolkemper, 2008), cytotoxic (Topçu et al., 2008) and antitumor (Fiore et al., 2012) activities. The major constituents of the aerial parts of the genus are flavonoids and triterpenoids (Topçu, 2006). This investigation of the chemical constituents of Salvia phlomoides was undertaken to investigate the flora of Algeria, and it led to the isolation of previously unreported oleanane triterpenoids 1, 2 and 3. Previously root material sourced in Spain was investigated and yielded a range of abietane diterpenoids (Hueso-Rodríguez et al., 1983; Rodriguez, 2003) and aerial parts yielded a range of 3,11- and 2,3 oxygenated lupane derivatives (Savona and Rodriguez, 1980; García-Alvarez et al., 1981). 2. Results and discussion Fractionation of the chloroform extract of the aerial parts of Salvia phlomoides led to the isolation of three unreported triterpenoids, compounds 1-3 (Fig. 1), α-amyrin and 5-hydroxy-7,4’-dimethoxyflavone. The EtOAc extract yielded caffeic acid and the flavonoids, apigenin, luteolin and apigenin-7-O-glycoside, all reported for the first time from this species. The HR-ESI spectrum of compound 1, isolated as a white solid, showed a [M+Na]+ ion at m/z = 481.36522, consistent with the molecular formula C30H50O3 for the compound. The IR spectrum showed a hydroxy group stretch at 3365cm-1. The 13C NMR spectrum in conjunction with the DEPT spectrum (Table 1) showed 30 signals including 8 upfield methyl group proton signals of a pentacyclic triterpenoid (Mahato and Kundu, 1994), two signals in the alkene region (δC130.2 and δC141.7) six fully substituted, eight methylene and six methine (including three oxymethine) carbons indicating an oleanane triterpenoid. A resonance at δC 56.2 was assigned as C-5 and it showed correlations in the HMBC spectrum with the 3H-23 (δH 1.03), 3H-24 (δH 0.84), oxymethine H-3 (δH 3.00, dd, J= 3.2, 9.6 Hz), 3H-25 (δH 1.17) and H-9 (δH 1.39) resonances. The H-3 resonance showed coupling in the COSY spectrum with the H-2 oxymethine resonance (δH 3.72, m), which, in turn, showed correlations in the HMBC spectrum with the C-4 (δC 39.5) and C-10 (δC 40.3) resonances. The COSY spectrum showed coupling between the H-9 and H-11 oxymethine resonance (δH 4.01, m) so the third hydroxy group was placed at C-11. The H-11 resonance showed a correlation in the HMBC spectrum with the methine C-13 (δC 37.7) resonance, which, in turn, showed a correlation with the 3H-27 (δH 0.75) and alkene H-19 (δH 4.86) resonances. The H-13 resonance (δH 2.43) showed correlations in the HMBC spectrum with the C-18 (δC 141.7), C-19 (δC 130.2) and C-17 (δC 34.5) resonances, the C-18 resonance showed correlations with the 3H-28 (δH1.01) resonance, and the H-19 (δH 4.86) resonance showed correlations with the 3H-29 (δH 0.95) and 3H-30 (δH 0.95) methyl group proton resonances. Thus the double bond was placed at C-18. The relative configuration of compound 1 was determined using the NOESY spectrum where correlations were seen between the H-3/3H-23, H-3/H-5; H-2/3H-24, H-2/3H-25, H-5/H-9, H-9/3H-27, H-11/3H-26, H-11/3H-25, H-13/3H-26, H-13/3H-28, H-19/H-12α and β, H-19/3H-29, H-19/3H-30, 3H-24/3H-25 resonances confirming that the hydroxy groups were at C-2α, C-3β and C-11α. The downfield chemical shift of H-1α was unusual. The HSQC spectrum clearly showed a correlation between the C-1 resonance (δC 49.7) and the H-1 resonances at δH 3.10 and δH 1.05. The unusually downfield H-1 resonance at δH 3.10 was seen to correlate with the 3H-25 (δH1.17) resonance in the NOESY spectrum and was thus assigned as H-1β. This assignment was confirmed by the two coupling constants (J=4.6,12.8 Hz), the 12.8 Hz coupling constant would be due to geminal coupling with H-1α and the 4.6 Hz coupling constant would be due to axial/equatorial coupling with H-2β. A model shows that H-1β lies directly between the oxygens of the hydroxy groups at C-2α and C-11. A literature search on the effect of a 11α-hydroxy group on the H-1β resonance showed that in 3β,11α,28-trihydroxyolean-18-ene, the two H-1 resonances occurred at δH 1.14 and δH 2.67 (Osorio et al., 2012), thus a hydroxy group at C-11α has a marked downfield effect on the H-1β resonance here, and the hydroxy group at C-2α in compound 1 causes an additional deshielding effect moving the resonance to the unusual downfield shift of δH 3.10. Thus compound 1 was identified as 2α,3β,11α-trihydroxyolean-18-ene. Compound 2 was isolated as a white solid. HR-ESI analysis indicated a [M+Na]+ peak at m/z = 523.37570, indicating a molecular formula of C32H52O4 for the compound. The IR spectrum displayed bands at 3368 and 1720 cm-1 ascribed to hydroxy and carbonyl stretches respectively. The NMR spectra were very similar to those of compound 1 except that the 13C NMR spectrum showed an extra carbon resonance at δC172.6 and nine methyl group carbon resonances. The 1H NMR spectrum showed an addition downfield methyl group proton resonance at δH 2.13, indicating that one of the hydroxy groups present in compound 1 had been converted to an acetate. The acetate was placed at C-3β as the H-3 resonance seen at δH 3.00, in compound 1 had shifted downfield to δH 4.50 (d, J=10.0 Hz) in compound 2. Coupling between the H-3α / H-2β (δH 3.81) / two H-1 (δH3.20, δH1.05) resonances was seen in the COSY spectrum. Thus compound 2 was identified as 3β-acetoxy,2α,11α-dihydroxyolean-18-ene. Compound 3 was obtained as a white solid, and the HR-ESI mass spectrum displayed an [M+Na]+ ion at m/z= 523.37570 corresponding to a molecular formula of C32H52O4 as for compound 2. The IR spectrum showed bands at 3461 and 1720 cm-1 characteristic of hydroxy group and carbonyl stretches respectively. The 13C and 1H NMR spectra again indicated the presence of a monoacetate with resonances at δC 172.6 and δC 21.7 and δH 2.06 and the resonance ascribed to H-2 in compound 1 shifted downfield to δH 5.01. The COSY spectrum showed coupling between the H-3 (δH 3.17), H-2 (δH 5.01) and two H-1 (δH 3.05 and 1.05) resonances confirming the structure to be 2α-acetoxy-3β,11α-dihydroxyolean-18-ene. Compounds 1-3 have not been reported previously. Analogous 2,3,11-hydroxylupenyl derivatives have been reported from Salvia phlomoides from Spain, and 2α,3β-dihydroxyolean-18-ene was reported from Salvia cabulica (Shazia et al., 2001) so it is not surprising that these oleanyl derivatives have now been found in the Algerian collection. Compounds 1-3 showed weak selective inhibitory effects in the NCI60 human tumour cell line screen at a single dose of 10µM, with compound 2 the most active. Compound 2 showed 38% growth inhibition against the RPMI-8226 leukaemia cell line, 36 and 34% against the non-small cell lung cancer NCI-H460 and NIC-H522 cell lines respectively, and30% inhibition of the colon cancer HT29 cell line. Full screening data is supplied in the Supplementary data. 3. Experimental section 3.1 General experimental procedures IR spectra were recorded using a Perkin-Elmer (2000 FTIR) spectrophotometer using KBr windows. 1H, 13C and 2D NMR spectra were recorded on a Bruker AVANCE III NMR spectrometer, operating at 500 MHz for 1H and 125 MHz for 13C, using standard experiments from the Bruker pulse programs library. Chemical shifts are reported in ppm () referencing the solvent signal (CDCl3 or CD3OD) as internal standard respect to TMS (0 ppm), and coupling constants (J) are measured in Hz. HR-ESIMS was performed on a Bruker MicroToF Mass Spectrometer, using an Agilent 1100 HPLC to introduce samples. Gravity column chromatography was performed using silica gel (Merck 230-400 mesh) packed 1 or 4 cm diameter columns. Compounds were visualized under UV light at 365nm, followed by spraying with 1% vanillin-H2SO4 spray reagent and heating. 3.2 Plant material: Aerial parts (leaves and flowers) of Salvia phlomoides were collected in April 2012 from Constantine in the east of Algeria (Coordinates: N 36°32’44.97’’ E 6°62’34.35’’, 680m a.m.s.l.) and authenticated by Prof Mohamed Kaabache, Department of Biology and Plant Ecology, University of Setif 1, Algeria. A voucher specimen (SP-125-5-06) has been deposited at the VARENBIOMOL Research Unit Herbarium, University of Constantine 1, Algeria. 3.3 Extraction and isolation: Aerial parts of Salvia phlomoides (1400g of flowers and leaves) were shade dried and macerated with MeOH/H2O (8/2, v/v) at room temperature for 3 x 48 hours, followed by filtration.  After filtration, the extracts were concentrated using a rotary evaporator and diluted with distilled water (500 ml, 25°C), agitated and left to stand overnight.  The resulting aqueous solution was extracted successively with petroleum ether, CHCl3, EtOAc and n-butanol.  The organic extracts were concentrated to obtain to yield four extracts: petroleum ether (0.24g), CHCl3 (6.0 g), EtOAc (6.0 g) and n-butanol (12.0 g).  The chloroform and ethyl acetate extracts were examined in this work. The chloroform extract was fractionated by column chromatography over silica gel 60 (Merck 230-400 mesh), with gradient elution using CH2Cl2/acetone with increasing polarity to yield 21 fractions of 100 cm3. Fraction 3 gave a yellow precipitate (110 mg) identified as 5-hydroxy-7,4’-dimethoxyflavone (Yang, et al., 1995), fraction 5 gave a white precipitate (50 mg) identified as α-amyrin (Chaturvedula and Prakash, 2013), fraction 14 gave compound 1 as a pure white precipitate (30 mg) which was separated by filtration. Fraction 9 was fractionated further using flash chromatography using a 1/1: CH2Cl2/EtOAc solvent mixture collecting 10 cm3 fractions. Sub-fractions 21 and 12, gave pure compounds 2 (24 mg) and 3 (27 mg) respectively. The ethyl acetate extract was fractionated by column chromatography over silica gel 60 (Merck 230-400 mesh), with gradient elution using CHCl3/acetone with increasing polarity to yield 16 fractions of 100 cm3. Fraction 5 was further separated to yield apigenin (230mg) (Alwahsh et al., 2015) and luteolin (155 mg) (Lee et al., 2013), fraction 9 yielded, after further purification, caffeic acid (35 mg) (Dürüst et al., 2001) and fraction 13 yielded apigenin-7-O-glucoside (56 mg) (Güvenalp et al., 2006). The structures of known compounds were confirmed by comparison of NMR data against literature values as referenced above. A detailed separation scheme is provided in the Supporting Information.  3.4 Compound screening: The anticancer activity of compounds 1-3 was evaluated at a single dose of 10µM against the NCI60 panel of human tumor cell line which is derived from nine cancer cell types : leukemia, lung, melanoma, colon, nervous system, ovary, renal, prostate and breast cancer according to the NCI protocol (Shoemaker, 2006). 4. Compound characterization 2α,3β,11α-trihydroxyolean-18-ene (1):white solid; IR (neat on KBr windows) νmax 3365cm-1; 1H NMR (CDCl3, 500 MHz) and 13C NMR (CDCl3, 125 MHz) data are given in Table 1; HR-ESIMS m/z 481.36522[M+Na]+ (calcd for C30H50O3Na,481.36576). 3β-acetoxy,2α,11α-dihydroxyolean-18-ene (2): white solid; IR (neat on KBr windows) νmax 3368 and 1720 cm-1; 1H NMR (CDCl3, 500 MHz) and 13C NMR (CDCl3, 125 MHz) data are given in Table 1; HR-ESIMS m/z 523.37570 [M]+ (calcd for C32H52O4Na, 523.376330). 2α-acetoxy-3β,11α-dihydroxyolean-18-ene (3): white solid; IR (neat on KBr windows) νmax 3461 and 1720 cm-1; 1H NMR (CDCl3, 500 MHz) and 13C NMR (CDCl3, 125 MHz) data are given in Table 1; HR-ESIMS m/z 523.37570 [M]+ (calcd for C32H52O4Na, 523.376330). Acknowledgments LB thanks the Natural Products Research Group, Department of Chemistry, University of Surrey, Guildford, United Kingdom, for hosting her to undertake this work and the Developmental Therapeutics Program (DTP) of the National Cancer Institute of the United States (U.S.A) for compound screening. The authors thank Colin Sparrow of the University of Oxford for MS analyses. Appendix A. Supplementary data The supplementary data associated with this article can be found in the online version, at http://dx.doi.orh/xxxx/j.phytol.201y.xx.xxx References Alwahsh, Mohamed Ali A., Melati Khairuddean, Wong Keng Chong, 2015. Chemical Constituents and Antioxidant Activity of Teucrium barbeyanum Aschers. Rec. Nat. Prod. 9:1, 159-163. Asadi, S., Ahmadiani, A., Esmaeili, M.A., Sonboli, A., Khodagholi, F., Ansari, N., 2010. In vitro antioxidant activities and an investigation of neuroprotection by six Salvia species from Iran: a comparative study. Food Chem. Toxicol. 48, 1341-1349. 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Structures of compounds 1, 2 and 3 isolated from Salvia phlomoides Table 1: NMR data of compounds1-3 in CDCl3 No 1 2 3 No 1 2 3 13C NMR data 1H NMR data 1 49.7 50.9 48.6 1α 1.05 m 1.05 m 1.05 m β 3.10 dd (J = 4.6, 12.8) 3.20 dd (J = 4.7, 12.9) 3.05 dd (J = 4.8, 11.6) 2 69.5 67.8 77.4 2 3.72 m 3.81 m 5.01 m 3 83.6 84.8 81.0 3 3.00 dd (J = 3.2, 9.6) 4.50 d (J =10.0) 3.17 dd (J = 6.4, 10. 3) 4 39.5 39.5 30.2 4 - - - 5 56.2 56.1 56.0 5 0.82 m 0.88 m 0.88 m 6 18.2 18.2 18.1 6α 1.58 m 1.51 m 1.53 m β 1.37 m 1.38 m 1.36 m 7 35.7 35.6 35.6 7α 1.35 m 1.32 m 1.32 m β 1.49 m 1.48 m 1.48 m 8 43.2 43.2 43.2 8 - - - 9 56.5 56.4 56.1 9 1.39 m 1.40 m 1.40 m 10 40.3 40.1 40.2 10 - - - 11 71.1 71.0 70.1 11 4.01 m 4.01 m 4.01 m 12 38.7 38.7 38.6 12α 1.29 m 1.30 m 1.30 m β 1.74 m 1.72 m 1.72 m 13 37.7 37.7 37.6 13 2.43 br d (W1/2=19.7) 2.42 br d (W1/2=19.8) 2.42 br d (W1/2=28.0) 14 43.0 43.0 43.0 14 - - - 15 27.7 27.7 27.7 15α 1.12 m 1.09 m 1.09 m β 1.70 m 1.69 m 1.69 m 16 37.5 37.5 37.5 16α 1.35 m 1.34 m 1.32 m β 1.43 m 1.45 m 1.48 m 17 34.5 34.5 34.5 17 - - - 18 141.7 141.6 141.7 18 - - - 19 130.2 130.2 130.2 19 4.86 br d (W1/2=4.1) 4.87 br d (W1/2=4.1) 4.85 br d (W1/2=4.5) 20 32.6 32.6 32.6 20 - - - 21 33.5 33.5 33.5 21α 1.33 m 1.32 m 1.32 m β 1.45 m 1.44 m 1.43 m 22 37.8 37.8 37.8 22α 1.35 m 1.34 m 1.35 m β 1.42 m 1.42 m 1.41 m 23 28.9 28.9 28.8 23 1.03 s 0.99 s 1.01 s 24 17.0 17.6 16.9 24 0.84 s 0.88 s 0.87 s 25 18.3 18.2 18.0 25 1.17 s 1.17 s 1.20 s 26 17.6 17.8 17.6 26 1.09 s 1.08 s 1.09 s 27 14.6 14.6 14.5 27 0.75 s 0.75 s 0.75 s 28 25.5 25.5 25.5 28 1.01 s 1.01 s 1.04 s 29 31.5 31.5 31.5 29 0.95 s 0.94 s 0.94 s 30 29.3 29.3 29.3 30 0.95 s 0.95 s 0.95 s Ac - 172.6 172.6 - - - - 21.4 21.7 Ac - 2.13 s 2.06 s 3-OH - - - 3-OH 2.17 d (J = 3.2 Hz) 2-OH - - - 2-OH 1.86 br s (W1/2=10.5Hz) *Values in parenthesis are J or W1/2 in Hz 8 1 11