A new monoterpene glucoside and complete assignments of dihydroflavonols of Pulicaria jaubertii: potential cytotoxic and blood pressure lowering activity

Abstract One new monoterpene glucoside and five dihydroflavonols were isolated for the first time from the aerial parts of Pulicaria jaubertii and identified as p-menthane-2-O-β-D-glucopyranoside [1], dihydroquercetin (taxifolin) [2], 7,3′-di-O-methyltaxifolin [3], 3′-O-methyltaxifolin [4], 7-O-methyltaxifolin (padmatin) [5] and 7-O-methyl-dihydrokampferol (7-O-methylaromadenderin) [6]. The structures of these compounds were unambiguously assigned on the basis of NMR spectroscopic data (1H, 13C, DEPT, HSQC, HMBC) and MS analysis. 2D-NMR methods required revision of assignments of H-6 and H-8 for dihydroflavonol compounds. Possible cytotoxic activity as well as blood pressure (BP) lowering activity were tested. The alcoholic extract showed cytotoxic activity against prostate carcinoma (PC-3), breast carcinoma (MCF-7) and hepatocellular carcinoma (HepG-2) human cell lines with IC50 19.1, 20.0 and 24.1 μg, respectively. The higher dose levels of the alcoholic extract significantly reduced normal BP of rats in a dose-dependent manner.


Introduction
Genus Pulicaria, belonging to the Asteraceae family, tribe Inuleae, consists of 100 species distributed in Europe, North Africa and Asia especially Yemen and Saudi Arabia and is known for its medicinal properties (Dubaie & El-Khulaidi 1993). Earlier phytochemical investigations on different Pulicaria species afforded several sesquiterpenoids (Stavri et al. 2008), caryophyllenes and caryophyllane derivatives (Marco et al. 1992), diterpenoids (Das et al. 2005), triterpenes (Eshbakova & Saidkhodzhaev 2001) and flavonoids (Christine et al. 2003;Williams et al. 2003). Pulicaria jaubertii [syn. Pulicaria orientalis Jaub. and Spach] is a fragrant perennial herb and it is traditionally used to treat inflammation, fever and as an insect repellent. Its essential oil composition as well as antimicrobial, antifungal, antimalarial and insecticidal properties were previously investigated (Dubaie & El-Khulaidi 2005;Al-Gabr et al. 2012;Fawzy et al. 2013). However, there have been no reports on the characterisation and isolation of the constituents of P. jaubertii. The present work describes the isolation and characterisation of one new [1] and five known compounds [2][3][4][5][6] from the aerial parts of the plant which have not been reported for P. jaubertii before. The cytotoxic activity of the alcoholic extract was tested against prostate carcinoma (PC-3), breast carcinoma (MCF-7) and hepatocellular carcinoma (HepG-2) human cell lines. The blood pressure modulating effect of the alcoholic extract was also evaluated in albino rats.

Results and discussion
Dried aerial parts of P. jaubertii were extracted with methanol and then fractionated with n-hexane, ethyl acetate and n-butanol. From the ethyl acetate extract and using combined chromatographic separations, one new [1] and five known compounds [2-6] were isolated ( Figure 1). Their structures were elucidated using physicochemical and spectral methods.
The new compound 1 was obtained as white powder. Its ESIMS showed a molecular ion peak at m/z 317 [M-H] − corresponding to the molecular formula C 16 H 30 O 6 . The IR spectrum showed a strong absorption band at 3340 cm −1 due to the hydroxyl functions in the molecule. 13 C NMR spectrum (Table S1) and DEPT experiment of 1 displayed the presence of one β-D-glucopyranosyl unit from the signals at δ 100.42 (C-1), 73.94 (C-2), 77.46 (C-3), 70.86 (C-4), 77.25 (C-5) and 61.82 (C-6) (Bradbury & Jenkins 1984) together with 10 carbon signals for the aglycon as follows: three methyl groups (δ 18.92, 19.97, 20.08), three methylene groups (δ 29.06, 29.32, 32.31) and four methines (δ 32.36, 35.91, 35.99, 75.36). The carbon signal at δ 75.36 indicated the attachment of one hydroxyl group to this carbon (Breitmaier & Voelter 1987). On the other hand, 1 H NMR spectrum of 1 (Table S1) revealed the presence of one methyl doublet at δ 0.82 having the integration of six protons assignable for Me-9 and 10 and one methyl doublet at δ 0.88 having the integration of three protons assignable for Me-7 (Laswell & Hufford 1977). Moreover, the one proton multiplet signal at δ 1.35 in the 1 H NMR spectrum of 1 was interpreted for H-8 while the six protons multiplet at δ 0.91-1.58 was assigned for three methylene groups (H-3, 5 and 6). The β-configuration of the glucopyranosyl unit was deduced from the coupling constant (7.7 Hz) of the anomeric proton doublet at δ 4.12 in the 1 H NMR spectrum of 1. The attachment of the β-glucosyl moiety to C-2 of the aglycon was based on the downfield shift of this carbon (δ 75.36) in addition to the long-range correlation observed in the HMBC spectrum between the anomeric proton (δ 4.12) and C-2 (δ 75.36). According to the above data, the structure of the aglycon of 1 is supposed to be one menthol isomer. This was confirmed by the long-range correlations observed in its HMBC spectrum. In fact, the occurrence of the long-range correlations between the methyl protons (H-7, δ 0.88 ppm) and the methine carbons (C-1, δ 35.91 ppm, C-2, δ 75.36 ppm and C-6, δ 29.32) suggests that the hydroxyl group should be present at position 2 instead of position 3 as in menthol. Additionally, in the 1 H-1 H COSY spectrum, correlations among the protons of a methine at δ 1.41 (H-1), an oxygenated methine at δ 3.79 (H-2) and a methyl at δ 0.88 (H-7) confirmed that, C-2 is the oxygenated carbon. Consequently, the structure of compound 1 was assigned as p-menthane-2-O-β-D-glucopyranoside which was isolated for the first time from nature.
Compounds [2][3][4][5][6] were obtained as colourless crystals except compound 3 was obtained as off-white amorphous powder. All compounds [2-6] gave an intense purple-pink colour when treated in methanol solution with granular magnesium and conc. HCl (Shinoda test), indicative of a flavonoid ring system (Dean 1963). IR absorptions of compounds 2-6 showed the same pattern, suggesting the presence of hydroxyl (3400-3460 cm −1 ) and carbonyl (1645-1655 cm −1 ) groups. In addition, their UV spectra recorded in methanol were nearly identical to each other and were in accordance with dihydroflavonols having band I as a shoulder at 328-332 nm on band II (290-292 nm). In the EIMS, a peak corresponding to [M] + was observed at m/z 304 showing the molecular formula of 2 to be C 15 H 12 O 7 . Compound 3 exhibited an [M] + peak at m/z 332, which was indicative of the molecular formula of C 17 H 16 O 7 . The EIMS of 4 and 5 showed the same molecular ions [M] + at m/z 318, 14 mass units more than that of 2, indicating 4 and 5 has an additional methyl group vs. 2 and appropriate for the molecular formula C 16 H 14 O 7 . Compound 6 exhibited an EIMS molecular ion peak at m/z 302 [M] + , which was indicative of the molecular formula of C 16 H 14 O 6 .
The 13 C NMR spectra showed that 2-6 were dihydroflavonols-type showing a numerous signals, particularly in the high field-region associated with the heterocyclic C ring. The chemical shifts of the C-2 and C-3 carbon atoms were characteristic of a dihydroflavonol-type. The 13 C NMR spectrum also showed that the B ring of 2-5 bore two ortho-hydroxyl groups from the carbon resonances at δ 144.34-147.81 (C-3′ and C-4′), confirmed 3′, 4′-dihydroxy B ring. While in 6, the two signals at δ 129.49 and 114.88 had intensities suggesting that they represent two carbons each, confirmed 4′-monohydroxy B ring. The remaining carbon signals were more or less consistent with the chemical shift values for the phloroglucinol type A ring and the catechol or p-substituted B ring of dihydroflavonol (Agrawal 1989). Furthermore, the 1 H and 13 C NMR spectra revealed the presence of two methoxyl groups in 3 and one methoxyl in each of 4, 5 and 6 (Tables S2 and S3). The location of the methoxyl groups was assigned on the bases of HMBC correlations, together with the shifts induced in the UV spectra by different reagents (see experimental) (Markham 1982). This allowed us to conclude that the two methoxyls of compound 3 were located at C-3′ and C-7 and that of compounds 4, 5 and 6 were located at C-3′, C-7 and C-7, respectively. Thus, compounds 2-6 were established as trans-dihydroquercetin (trans-taxifolin), 7, 3′-di-O-methyltaxifolin, 3′-O-methyltaxifolin, 7-O-methyltaxifolin (padmatin) and 7-O-methyl-dihydrokampferol (7-O-methylaromadenderin), respectively (Herz et al. 1972;Balza & Towers 1984;Grande et al. 1985;Agrawal 1989;Harborne 1994;Marco et al. 1994;Wang et al. 2012).

Cytotoxic activity
The cytotoxic activity of the alcoholic extract of P. jaubertii was investigated against three human tumor cell lines, PC-3, MCF-7 and HepG-2 using cell viability assay method. As shown in Figure S1 and Table S4, the alcoholic extract revealed different cytotoxic activities towards the three human cancer cell lines investigated. In general, a concentration-dependent decrease in the survival of the three tumor cell lines was observed. At a concentration of 1.56 μg/mL, the alcoholic extract did not considerably affect the viability of the three human tumor cell lines compared with untreated control cells. The cell survival after treatment with alcoholic extract was more than 90%. At a concentration of 50 μg/mL, the alcoholic extract exhibited strong cytotoxicity towards PC-3 cells. Cell viability was lower than 19%. The IC 50 values for the alcoholic extract against these three cell lines were 19.1, 20 and 24.1 μg/mL, respectively. PC-3 cells were more sensitive than the other cell lines followed by MCF-7 cells. HepG-2 cells were relatively less sensitive than the other cell lines.

Blood pressure lowering effect
As shown in Figure S2, SBP of the control group was 115 ± 8.26 mm Hg. Following administration of the extract in the lowest dose level, a statistically insignificant decrease in SBP to 105 ± 10.6 mm Hg was observed. However, the higher dose levels significantly reduced SBP in a dose-dependent manner, down to 100 ± 5.5 mm Hg and 90 ± 7.4 mm Hg for 10 and 20 mg/kg, respectively. The decrease in SBP was transient and returned to normal within two min. No significant change in heart rate was observed at the dose levels used (data not shown).

Material and apparatus
UV spectra were determined with a Shimadzu UV-1650PC spectrophotometer; IR spectra were carried out on a Nicolet 205 FT IR spectrometer connected to a Hewlett-Packard Color Pro. Plotter. The 1 H-and 13 C-NMR measurements were obtained with a Bruker Avance spectrometer operating at 600 MHz (for 1 H) and 150 MHz (for 13 C), a Bruker BioSpin AG operating at 500 MHz (for 1 H) and 125 MHz (for 13 C) and a Bruker Avance III NMR spectrometer operating at 400 MHz (for 1 H) and 100 MHz (for 13 C) in DMSO-d 6 solution, and chemical shifts were expressed in δ (ppm) with reference to TMS and coupling constant (J) in Hertz. 13 C multiplicities were determined by the DEPT pulse sequence (135°). COSY, HMBC and HSQC NMR experiments were carried out using a Bruker AV-600, a Bruker BioSpin AG 500 and a Bruker AV-400 III spectrometers. EIMS was carried on Scan EIMS-TIC, VG-ZAB-HF, and X-mass (158.64, 800.00) mass spectrometer (VG Analytical, Inc.). Si gel (Si gel 60, Merck), was used for open column chromatography. Solid phase extraction was performed on SPE-C 18 cartridges (Strata columns). TLC was carried out on precoated silica gel 60 F254 (Merck) plates. Developed chromatograms were visualised by spraying with 1% vanillin-H 2 SO 4 , followed by heating at 100 for 5 min.

Plant material
The aerial parts of P. jaubertii were collected from Aljar region of Hajja-Yemen in August 2012, and were kindly identified by Dr Abd El-Rahman Saeed Aldabee, Professor of Plant Taxonomy, Faculty of Science, Sana`a University, Yemen. A voucher specimen {P-01} has been deposited in the Pharmacognosy Department, Faculty of Pharmacy, Al-Azhar University, Cairo, Egypt.

Extraction and isolation
The dried aerial parts of P. jaubertii (1.5 kg) were extracted with MeOH three times to yield 85 g of a dark solid extract, which was then suspended in water (500 ml) and successively partitioned with n-hexane, ethyl acetate and n-butanol to obtain n-hexane (30 g), ethyl acetate (12 g) and n-butanol (8 g) extracts after evaporating the solvent. The ethyl acetate extract was chromatographed on a silica gel column and eluted with methylene chloride 100% to obtain four fractions of A (40 mg), B (150 mg), C (200 mg) and D (750 mg), followed by elution with ethyl acetate 100% to obtain five fractions of E (1.1 g), F (660 mg), G (490 mg), H (510 mg) and I (1.5 g). The C fraction (200 mg) was repeatedly applied to Sephadex LH-20 eluted with MeOH to give compound 3 (37 mg). Fraction D (750 mg) was applied to Sephadex LH-20 eluted with MeOH to give two sub fractions of D-1 (215 mg) and D-2 (60 mg). The sub fraction D-1 was chromatographed on a silica gel column and eluted with pet. ether-ethyl acetate 80:20 to afford compound 6 (14 mg). Fraction E (1.1 g) was applied on a Sephadex LH-20 column eluted with MeOH to gives two sub fractions of E-1 (890 mg) and E-2 (58 mg). Fraction E-1 was subjected to solid-phase extraction using 10:90-80:20 acetonitrile: water system to give three subfractions of E-1a (110 mg), E-1b (87 mg) and E-1c (220 mg). Subfractions E-1a and E-1b were finally purified on a Sephadex LH-20 column eluted with MeOH to give compound 4 (80 mg) and compound 5 (60 mg), respectively. Fraction G (490 mg) was applied on a Sephadex LH-20 column eluted with MeOH to gives two sub fractions of G-1 (350 mg) and G-2 (17 mg). The sub fraction G-1 was chromatographed on a silica gel column and eluted with pet. ether-ethyl acetate 65:35 to afford G-1a (37 mg) and H-1b (135 mg). Fraction G-1b (135 mg) was further chromatographed on a silica gel column and eluted with pet. ether-ethyl acetate 70:30 to afford compound 2 (45 mg). Fraction I (1.5 g) was applied to Si gel CC and eluted with pet. ether-ethyl acetate 50:50-20:80 to gives two sub-fractions of I-1 (150 mg) and I-2 (700 mg). The sub-fraction I-2 was applied to Sephadex LH-20 eluted with MeOH to give two fractions of I-2a (40 mg) and I-2b (185 mg). Fraction I-2a was subjected to solid-phase extraction (C18 column) eluted with 100% water-80% water in acetonitrile to afford compound 1 (6 mg).

Cytotoxicity assays
The cytotoxicity of the alcoholic extract was tested against three human tumor cell lines; prostate carcinoma (PC-3), breast carcinoma (MCF-7) and hepatocellular carcinoma (HepG-2) cell lines. The cells were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA). The cells were grown on Roswell Park Memorial Institute (RPMI) 1640 medium (Nissui Pharm. Co., Ltd., Tokyo, Japan) supplemented with 10% inactivated fetal calf serum and 50μg/mL gentamycin. The cells were maintained at 37°C in a humidified atmosphere with 5% CO 2 and were subcultured two to three times a week. The cytotoxic activity was determined using cell viability assay method as described previously (Mosmann 1983;Gangadevi & Muthumary 2007). The experiments were performed in triplicates and the percentage of cell viability was calculated as the mean absorbance of control cells/mean absorbance of treated cells. Concentration-response curves were prepared and the IC 50 values were determined.

Evaluation of blood pressure lowering effect
Adult male albino Sprague-Dawley rats weighing 150-200 g were used in our experiments. They were purchased from the animal facility of the pharmacology department, College of Pharmacy, King Abdul-Aziz University, Jeddah, KSA. Animals were housed in cages kept under constant environmental and nutritional conditions throughout the period of investigation. They were allowed a free access to water and diet consisting of standard pellet chow. The study was carried out according to The European Communities Council Directive of 1986 (86/609/EEC) and approved by the Ethical Committee for Animal Experimentation at the Faculty of Pharmacy, Umm Al Qura University, Makkah, KSA.
Systolic blood pressure (SBP) was measured in urethane anaesthetised animals (1.3 g/kg i.p.) directly from the left common carotid artery according to the method of Burden et al. (1979), via a polyethylene cannula attached to a pressure transducer (APT300, Hugo Sachs Elektronik, Germany) connected to an oscillographic recorder (Harvard Apparatus, UK). The right jugular vein was similarly cannulated for intravenous injection of the extract under investigation. The extract was freshly reconstituted and injected intravenously in the right jugular vein. Three dose levels of the alcoholic extract were used; 5, 10 and 20 mg/Kg. The experiment was repeated in six animals. A control group composed of six rats was injected with the solvent in the same volume as the treated group. Changes in SBP were recorded and read directly from the tracing that was calibrated at the end of each experiment.

Statistical analysis (Woodson 1987)
All data were expressed as mean ± SE. Student's t-test was applied for detecting the significance of difference between the values of the treated groups and the control. Differences were considered significant when p value was <0.05.

Conclusion
The present study has identified the isolation and characterisation of a new monoterpene glucoside along with five known dihydroflavonol for the first time from the aerial parts of P. jaubertii. It may be also concluded that the standardised P. jaubertii extract significantly reduced SBP in a dose-dependent manner without significant change in heart rate at the dose levels used. Further investigations using the isolated fractions of the extract are now being carried out to elucidate the active principle(s) that owe this hemodynamic effect.