Acylated flavonol glycosides from Tagetes minuta with antibacterial activity

Wild marigold (Tagetes minuta), a flowering plant of the family Asteraceae contains compounds of pharmaceutical and nutritional importance especially essential oils and flavonols. Identification, characterization of flavonols and determination of their antibacterial activity were major objectives of the current study. The isolation and purification of flavonols was accomplished using chromatographic techniques while structural elucidation was completed by LC–MS and NMR spectroscopy. The extracts and purified compounds were tested against various bacterial strains for antibacterial activity. A total of 19 flavonols were isolated from this species. Of these, 17 were of butanol and two of ethyl acetate extracts. Based on the concentration and purity, eight potential flavonols were selected and structurally elucidated. Four flavonols, 6-hydroxyquercetin 7-O-β-(6′′-galloylglucopyranoside; 2), 6-hydroxykaempferol 7-O-β-glucopyranoside (5), 6-hydroxykaempferol 7-O-β-(6′′-galloylglucopyranoside; 7), 6-hydroxyquercetin 7-O-β-(6′′-caffeoylglucopyranoside; 9), were identified for the first time from T. minuta. Butanol and ethyl acetate extracts of flowers and seeds showed significant antibacterial activity against Micrococcus leteus, Staphylococcus aureus, Bacillus subtilis, and Pseudomonas pikettii. Among the isolated flavonols only 1, 2, and 18 were found to possess significant antibacterial activity against M. luteus. The extracts and purified flavonols from T. minuta can be potential candidates for antibacterial drug discovery and support to ethnopharmacological use.


Introduction
The genus Tagetes includes 56 species (Soule, 1993), widely grown all over the world for multipurpose uses including ornamental, medicinal, cultural, nutritional and therapeutics (Vasudevan et al., 1997;Shahzadi et al., 2010). Tagetes minuta (wild marigold) is a potential medicinal plant of the family Asteraceae and is widely grown from temperate to tropical regions of the world in a wider range of climatic conditions (altitude range from 3000 to 11000 feet).
Tagetes minuta is reported to contain a number of chemical and biochemical compounds of high pharmaceutical and nutritional value (Shahzadi et al., 2010). The prominent classes of compounds present in this species are essential oils and flavonols. Majority of the studies focused on the isolation and characterization of essential oils of T. minuta (Singh et al., 1992;Tankeu et al., 2013). Efforts toward the identification, isolation, and characterization of flavonols in T. minuta have been marginal.
Flavonols are secondary metabolites which represent a class of flavonoids and are widely distributed in almost all plant groups like angiosperms, gymnosperms, mosses, liverworts, and ferns. More than 393 flavonols (Vetschera and Wollenweber, 2006) and over 1331 flavonol O-glycosides (Williams, 2006) were reported with complete structural elucidation. Flavonols and flavones accumulate in the epidermal cells consequent to wounding, pathogenic attack, nutrient deficiency, temperature changes, ozone irradiation, UV protection and copigmentation in flowers and fruits (Flint et al., 1985;Koes et al., 1994;Shirley, 1996;Smith and Markham, 1998;Simmonds, 2003). These also enhanced wine color through copigmentation with anthocyanins (Baranac et al., 1997;Boulton, 2001). Flavonols are natural products and preparations containing these compounds as the principal physiologically active constituents have been employed in curing many diseases in human since ages (Cushnie and Lamb, 2005). Several plant-derived flavonols have been reported to possess antibacterial activity against some super bugs, which are becoming common causes of infections in the acute and long-term care units in hospitals (Xu and Lee, 2001;Chan et al., 2014).

Plant Material
Tagetes minuta L plant was collected during August-October, 2010 for chemical analysis from district Abbottabad, Pakistan. The collected plants were first identified by a taxonomist and a voucher specimen was deposited (IS/ATD/H-03) at the herbarium of the Botany Department of Post-Graduate College, Abbottabad, Pakistan. The plant material was separated and divided into three different portions; (A) flowers and seeds (5.2 kg), (B) roots (3.5 kg), and (C) leaves and stems (10 kg). These portions were air dried in shade and were crushed to fine powder by locally built grinder.

Extraction and Fractionation
Powder of all plant parts were extracted at room temperature by maceration with methanol (5L X 3) for 72 h. The methanol extracts were concentrated under reduced pressure at 30 • C to give dry marc of 356 g (A), 106.4 g (B), and 461 g (C). These crude extracts were separately suspended in distilled H 2 O and subsequently extracted with n-hexane, chloroform, ethyl acetate and n-butanol. Each fraction was dried over anhydrous sodium sulfate and evaporated to yield dried fraction HA (n-hexane, 105 g), fraction CA (chloroform, 39.3 g), fraction EA (ethyl acetate, 21.4 g), fraction BA (butanol, 53.6 g), and WA (aqueous, 120.7 g) from flower and seeds extract and fractions, HB (nhexane, 10.7 g), CB (chloroform, 3.8 g), EB (ethyl acetate, 1.3 g), BB (butanol, 1 g), and WB (aqueous, 67.6 g) from roots extract and fractions HC (chloroform, 146.4 g), CC (chloroform, 118.6 g), EC (ethyl acetate, 16.9 g), BC (butanol, 17.9 g), and WC (aqueous, 131.6 g) from leaves and stem extract. The crude extracts were defatted by n-hexane therefore fractions HA, HB, HC and in addition aqueous fractions were also not used for further analysis.
Fractions EA (ethyl acetate, 21.4 g) and BA (butanol, 53.6 g) were separately applied to a Sephadex LH-20 column (Pharmacia, 5 cm × 50 cm) and eluted with methanol. Eighteen fractions (∼200 mL each) were collected and evaporated to dryness under reduced pressure. Fractions 1-10 containing flavonol constituents were combined, owing to their TLC similarities, and subsequently purified by semi preparative HPLC.

Isolation of Flavonols -Preparative HPLC
Various chloroform, ethyl acetate and butanol fractions were analyzed by high performance liquid chromatography (HPLC). Samples were filtered through a 0.45 μm Millipore membrane filterprior to injection.
The butanol and ethyl acetate fractions of flowers and seeds were subjected to preparative HPLC by (A) H 2 O-TFA (0.5%) and (B) Acetonitril-TFA (0.5%) solvents for the isolation of flavonoids using a Gilson 305/306 pump equipped with C 18 reversed phase column [ODS-Hypersil column (25 cm × 2.2 cm, 5 μm)] coupled to a multidiode array detector (HP-1040 A; Supelco, Bellefonte, PA, USA). The elution profile consisted of a linear gradient from 10% B to 100% B for 40 min, isocratic elution (100% B) for the next 3 min, followed by the linear gradient from 100% B to 10% B for 5 min. The flow rate was 15 mL/min, and aliquots of 500 μL were injected.

Reversed Phase Analytical HPLC-DAD
The Agilent 1100 HPLC system was equipped with a HP 1050 diode-array detector and a 200 mm × 4.6 mm, 5 μm ODS Hypersil column (Supelco, Bellefonte, USA). The elution system was binary, with A, water (0.5% TFA), and B, acetonitrile (0.5% TFA) solvents. The elution profile for HPLC consisted of initial conditions with 90% A and 10% B followed by linear gradient elution for the next 18 min to 20% B and the subsequent linear gradient conditions: 18-26 min (to 23% B), 26-30 min (to 28% B), 30-40 min (to 100% B), isocratic elution 40-43 min (100% B), and final linear gradient elution 43-48 min (to 10% B). The flow rate was 1.0 mL/min, and aliquots of 20 μL were injected with an Agilent 1100 series micro auto sampler. The UV absorption spectra were recorded on-line during HPLC analysis over the wavelength range of 280-360 nm in steps of 2 nm.

Turbidity Standard, Growth Media, and Culture Conditions
To compare the turbidity of bacterial culture McFarland (0.5 BaSO 4 ) solution was used. The standard was prepared by adding 0.5 ml of 1% solution (w/v) of anhydrous BaCl 2 to 99.5 ml of 1% solution (v/v) of H 2 SO 4 . Barium sulfate turbidity (4-6 ml) was taken in screw capped test tube and was used to compare the turbidity of bacteria. Nutrient broth medium for bacterial growth consisted of peptone (5 g/L), sodium chloride (5 g/L), starch (1 g/L), and agar (10 g/L; Oxoid, England). The nutrient agar plating medium consisted of plury peptone (5 g/L), beef extract (3 g/L), sodium chloride (8 g/L), and agar (15 g/L; Britanialab). Both media were prepared according to manufacturer's instructions in distilled water and sterilized in autoclave at 121 • C for 15 min. The bacterial cultures of selected strains were inoculated individually and incubated in a shaker at 150 rpm for overnight at 37 • C.

Antibacterial Activity
The agar well-diffusion method (Bibi et al., 2011) analyzed the samples for antibacterial activity. The autoclaved nutrient agar media was allowed to cool at 45 • C and 25 mL was poured in each petri plate near flame. These petri plates were incubated overnight at 37 • C in an incubator to avoid contamination. The wells were made by 6 mm sterile metallic borer at 24 mm distance from each other. Extracts and isolates were analyzed at 3 mg/mL in dimethyl sulfoxide (DMSO; Sigma-Aldrich, USA). Ampicillin (Sigma-Aldrich, USA, A2804) was used as standard antibiotic (positive control), up to 50 μg/mL and pure DMSO was used as negative control. Each well was filled with 20 μL of plant extract (60 μg), positive (Ampicillin, 20 μg) and negative (DMSO) control and labeled at the back of petri dishes. The incubation of sample plates was at 37 • C in incubator for 24 h and all cultures were prepared in triplicate. After 24 h of incubation period, diameter of clear zones around each well was measured, showing no bacterial growth. The mean zone of inhibition was calculated with standard deviation procedure.

Statistical Analysis
The experiments were conducted in duplicate and all the analyses were done in triplicate. One way ANOVA followed by the Duncan's multiple-range test was used to determine significant (p < 0.05) differences among different extracts of plant material. The computer software SPSS 16.0 (SPSS, Inc., Chicago, IL, USA, package) carried out statistical analyses. The data was expressed as mean ± standard deviation (SD).

Results and Discussion
The HPLC profiles revealed that the butanol fractions contained relatively high quantities of flavonoids as compared to the ethyl acetate fractions, while non-flavonoids compounds were identified in chloroform fractions. The roots, leaves, and stem extract profiles were not of high quality and therefore were not used for further spectroscopic analysis. The HPLC profiles of the butanol fraction (flowers and seeds) showed 17, while the ethyl acetate fraction of flowers and seeds showed two major flavonols ( Table 1). The absorption maxima in the UV spectra of these compounds were recorded online during HPLC analysis in the 320-360 nm region indicating flavonol 7-O-glycosides based on quercetagetin, 6-hydroxy kaempferol, quercetin, and patuletin aglycones (Harborne et al., 1991;Llorach et al., 2003;Veitch et al., 2011).
The sugar region of the 1D 1 H NMR spectrum of 1 showed the presence of one sugar unit by the single anomeric proton signal at 5.14 ppm (J = 7.4 Hz). In the COSY spectrum of this proton the observed crosspeak at 5.14/3.71 and the following crosspeaks were in accordance with seven sugar protons, which indicated that this sugar unit was a hexosyl. The corresponding crosspeaks in the HSQC spectrum permitted the assignment of H-2 , H-3 , H-4 , H-5 , H-6A and H-6B , and the chemical shifts and coupling constants of 1 were in accordance with β-glucopyranosyl (Harborne et al., 1991;Parejo et al., 2005). A cross peak at δ 5.14/152.5 (H-1 /C-7) in the HMBC spectrum confirmed the connection point of the sugar unit to be in the seven-position of the aglycone (Harborne et al., 1991). The molecular mass at m/z 481.0963 in the ESI high resolution mass spectrum of 1 was in accordance with 6-hydroxyquercetin 7-Oβ-glucopyranoside (C 21 H 20 O 13 + H + ; calc: 481.0982).
The chemical shift values in 1D 1 H NMR spectrum of 7 were similar to those of 5. It showed a singlet at 6.96 ppm (H-8) and a 4H ABXY system of two doublets at 7.9 ppm (d, 8.8 Hz; H-2 and H-6 ) and 6.92 ppm (d 8.9 Hz; were in accordance with the flavonol, 6hydroxykaempferol ( Table 2). The ESI-MS spectra showed the fragment ion due to cleavage of the glycosidic bond, which suggested the presence of 6-hydroxykaempferol (m/z 303.0485). Some of the NMR signals were missing or very weak, but the singlet at 7.15 ppm of H-2 and H-6 in the 1D 1 H NMR spectrum of 7 was in accordance with a galloyl unit (Brakat et al., 1999). The molecular mass at m/z 617.1137 in the high-resolution ESI-MS spectrum was in accordance with 6-hydroxykaempferol 7-O-β-(6 -galloylglucopyranoside; C 28 H 24 O 16 + H + ; calc: 617.1143).

Antibacterial Activity
The antibacterial activity of crude, aqueous, ethyl acetate, and n-butanol extracts of different parts (flowers and seeds, roots, leaves, and stems) of T. minuta were tested against gram positive (M. leteus, S. aureus, B. subtilis) and gram negative (P. pikettii, E. coli, S. setubal) bacteria ( Table 4). The isolated flavonols 1, 2, 5, 6, 7, 9, 18, and 19 were also subjected to antibacterial screening. All the samples showed significant antibacterial activity while these samples were inactive against E. coli and S. setubal. Activity comparison of three parts of plant indicated that flowers and seeds extract have statistically significant impact. Besides the extracts, only the isolated flavonols 1, 2 of butanol and 18 of ethyl acetate extract of flowers and seeds showed statistically significant (p < 0.05) activity against M. luteus. The crude extracts were statistically significant against gram positive (B. subtilis and S. aureus) and gram negative bacteria (P. pikettii). This study indicated that extracts and isolated flavonols of T. minuta have a broad spectrum of antibacterial activity.
The glycosilated and acylated flavonols of 6-hydroxyquercetin were strongly active in contrast to non-glycosilated flavonol. The results were in agreement with previous work of preliminary screening of the leaf extracts of C. tergemina displayed antibacterial activity against M. luteus, B. cereus, methicillinresistant S. aureus containing acylated flavonol glycosides (Chew et al., 2011;Chan et al., 2014). The antimicrobial effect of crude flavonoids extracts has been reported in leaves of T. minuta (Tereschuk et al., 1997(Tereschuk et al., , 2004. The antibacterial activity of essential oil containing dihydrotagetone and ocimene has also previously been reported in T. minuta against Gram (+) and Gram (-) bacteria (Senatore et al., 2004). In this study antibacterial activity of plant extracts and purified acylated flavonol glycosides (1, 2, and 18) was reported for the first time in T. minuta.

Conclusion
The extracts and purified compounds of plant were considered as suitable candidates for antibacterial drug discovery and support ethnopharmacological use, and its economical properties for commercialization of T. minuta. Based on these findings, we envision the discovery of new antibacterial agent from natural sources (plants) that will help to minimize the adverse effects of synthetic drugs.