Triterpenes and phenolic compounds isolated from the aerial parts of Herissantia tiubae and evaluation of 5,4′,-dihydroxy-3,6,7,8,3′-pentamethoxyflavone as a modulator of bacterial drug resistance

Abstract

In our continuous study of the Malvaceae family, we describe here the isolation of four triterpenes (frideline, lupeol, cycloartenol, and cycloeucalenol), a steroid (β-sitosterol), and four phenolic compounds from the aerial parts of Herissantia tiubae (K. Schum.) Brizicky (i.e., a benzoic acid derivative, a coumarin and two flavonoids, kaempferol 7-O-α-l-rhamnopyranoside and 4′,5-dihydroxy-3,6,7,8,3′-pentamethoxyflavone). The structural identification of the compounds was made by comparison with literature data and spectral analysis, including two-dimensional NMR techniques. These compounds are reported here for the first time in genus Herissantia. The pentamethoxyflavone was assayed against strains of Staphylococcus aureus possessing efflux mechanisms of resistance to norfloxacin, tetracycline, and erythromycin. Although the compound did not display relevant antibacterial activity (MIC ≥ 512 μg/mL), it modulated the activity of antibiotics, i.e., in combination with antibiotics (at 128 μg/mL), a two-fold reduction in the MIC values for tetracycline and erythromycin, a 32-fold reduction in the MIC for norfloxacin were observed.

Keywords::

• Malvaceae
• flavonoids
• antibacterial activity

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Corrigendum

Introduction

According to the new phylogenetic circumscription, the Malvaceae family consists of about 243 genera and 4225 species (Stevens, 2003), which are widespread in many regions of the world, particularly in tropical areas, mainly in South America (Heywood, 1993). Species of Malvaceae are used in folk medicine for many applications, such as a diuretic, in the treatment of rheumatism, gastrointestinal disorders (Schultz, 1968; Yesilada & Gürbüz, 2002), snakebites (Otero et al., 2000) and asthma (Franzotti et al., 2000). Anti-inflammatory and antinociceptive effects were also investigated (Venkatesh et al., 1999). Phytochemical investigations of Malvaceae species have described the isolation of fatty acids (Carmody et al., 1945; Vickery, 1980; Schimid & Patterson, 1988; Nakatani et al., 1986), essential oils (Ames & Macleod, 1990), sesquiterpenelactones (Sharma & Ahmad, 1989), triterpenes (Ahmed et al., 1990) and flavonoids (Silva et al., 2005a, 2005b), among many other compounds.

The genus Herissantia comprises six species, restricted to the tropical America with the major occurrence in Mexico, Antilles, and South America. Only Herissantia crispa L. (Brizicky) is widespread, occurring from the United States to Argentina. Herissantia tiubae (K. Schum) Brizicky, commonly known as mela bode, consists of a small shrub, found in the Brazilian northeast, mainly between the states of Bahia and Pernambuco (Corrêa, 1978). In previous publications concerning Herissantia tiubae, we described the isolation and structural identification of four polyoxygenated flavonoids (Silva et al., 2005a), to which has been attributed a broad spectrum of biological activity (Chen et al., 1997). In a different study we isolated two flavonol glycosides, kaempferol 3,7-di-O-α-l-rhamnopyranoside and kaempferol 3-O-β-D-(6′′-E-p-coumaroyl) glucoside (Silva et al., 2005b). Kaempferol 3,7-di-O-α-l-rhamnopyranoside was subjected to preliminary pharmacological tests, showing a relaxing effect over superior mesenteric artery (Silva et al., 2005b), which suggested a possible cardiovascular effect. Cycloeucalenol and cycloartenol, previously isolated from H. tiubae, showed a relaxant effect in the guinea-pig ileum (Gomes et al., 2005).

Efflux pumps are integral proteins of bacterial membrane accounting for much of bacterial resistance since they extrude antibiotics from the cell (Piddock, 2006). Resistance modifying agents are compounds that potentiate the activity of an antibiotic against resistance strains, and some of these agents may act as an inhibitor of efflux pumps (EPIs). Plants provide a rich source of EPIs and several compounds have been identified as potent inhibitors (Gibbons, 2004). In this work, we evaluated 4′,5-dihydroxy-3,6,7,8,3′-pentamethoxyflavone as modulator of antibiotic resistance using strains of Staphylococcus aureus possessing efflux pumps.

Materials and methods

Equipment

NMR spectra (¹H, ¹³C, HMQC, HMBC, COSY) were run on a Mercury-Varian instrument operating at 200 MHz and 50 MHz for ¹H and ¹³C, respectively, and also on a Brucker-AC operating at 300 (¹H) and 75 MHz (¹³C). The NMR data were measured in CDCl₃, CD₃OD and pyridine-d₆, and chemical shifts were expressed in parts per million (ppm) with reference to the solvent signal. Chromatography columns were used with silica gel (Merck) and Sephadex LH-20 (Pharmacia, Uppsala, Sweden). TLC was preformed with silica gel 60 F₂₅₄ (Merck); spots were visualized under UV light (244 and 366 nm), exposure to iodine vapor and by spraying with 5% FeCl₃ reagent (for detection of glycosyl flavonoids).

Plant material

The aerial parts were collected in January 2004 in the county of Juazeirinho, Paraíba state, Brazil. It was identified by Maria de Fátima Agra of the Universidade Federal da Paraíba. A voucher specimen (no. 2434) is deposited in the Herbarium Lauro Pires Xavier-JPB at the same university.

Extraction and isolation

Dried and ground aerial parts of H. tiubae (5000 g) were extracted with ethanol (3 X 10 L, 72 h each) and the crude extract was concentrated to dryness under reduced pressure to yield 190 g of a dark green residue. This material was partitioned with hexane, chloroform, ethyl acetate, and butanol. The hexane extract (20 g) was chromatographed on silica gel and eluted with hexane, gradually increasing the polarity with CHCl₃ and then MeOH, yielding 10 major fractions after analysis by TLC (SiO₂, hexane with increasing CHCl₃, iodine vapor). Fraction 3 was also subjected to chromatography over silica gel, using hexane, CHCl₃ and MeOH, resulting in 88 fractions, also combined by TLC analysis. Subfractions 05-13 and 14-57 led to isolation of compounds 1 and 2. Fraction 6, after two sucessive chromatographies on silica gel, yielded compounds 3, 4, and 5.

The CHCl₃ phase (27 g) was chromatographed over silica gel 60 and eluted with hexane, CHCl₃ and MeOH with increasing polarity, resulting in 100 fractions, which were combined after analysis by TLC (SiO₂, hexane:CHCl₃ 9:1, iodine vapor). Fractions 50-55 were subjected to repeated chromatographic procedures on silica gel 60, using hexane, CHCl₃ and MeOH, yielding compounds 6, 7, and 8, which were purified after recrystallization with hexane:CHCl₃ (9:1). Ethyl acetate extract (1.5 g) was applied to a Sephadex LH-20 column (Pharmacia, Uppsala-Sweden) with MeOH as eluent, where fractions of 20 mL were obtained and combined after analysis through TLC (SiO₂, CHCl₃:MeOH 1:1, FeCl₃ 5%). Fractions 13/16 were recrystallized with MeOH furnishing compound 9.

Bacterial strains

The strains of S. aureus used were SA-1199B, which overexpresses the norA gene encoding the NorA fluoroquinolones (and other drugs) efflux protein (Kaatz et al., 1993; Kaatz & Seo, 1995), RN4220 harboring plasmid pUL5054, which carries the gene encoding the MsrA macrolide efflux protein (Ross et al., 1989), and IS-58, which possesses the TetK tetracycline efflux protein (Gibbons & Udo, 2000). All strains were maintained on blood agar base (Difco) slants and, prior to use (assay), the cells were grown overnight at 37°C in brain heart infusion broth (BHI, Difco).

Antibiotics and 4′,5-dihydroxy-3,6,7,8,3′-pentamethoxyflavone

Norfloxacin, erythromycin and tetracycline were obtained from their respective manufacturers. The stock solutions of the antibiotics were prepared according to CLSI guidelines (2005). The stock solution of 4′,5-dihydroxy-3,6,7,8,3′-pentamethoxyflavone was prepared in DMSO, and its highest concentration remaining after dilution into broth (4%) caused no inhibition of bacterial growth.

Drug susceptibility testing

The minimum inhibitory concentrations (MICs) of the antibiotics and 4′,5-dihydroxy-3,6,7,8,3′-pentamethoxyflavone were determined in BHI by the microdilution assay using a suspension of ca. 10⁵ cfu/mL and a drug concentration range of 256 to 0.5 μg/mL (two-fold serial dilutions). The MIC is defined as the lowest concentration at which no growth is observed. For the evaluation of 4′,5-dihydroxy-3,6,7,8,3′-pentamethoxyflavone as a modulator of antibiotic resistance, the MICs of the antibiotics were determined in the presence of the compound at a sub-inhibitory concentration.

Constituents isolated from H. tiubae.

Results and discussion

The structure identification of compounds 1-7 was based on spectral analysis and also by comparison with literature data, which allowed their assignments as friedeline (1) (Cheung & Willianson, 1969), lupeol (2) (Vanderlei, 1985), cycloeucalenol (3) (Akihisa et al., 1989), cycloartenol (4) (Emmons et al., 1989), β-sitosterol (5) (Kojima et al., 1990), p-methoxybenzoic acid (6) (Marques et al., 1998) and 6,7-dimethoxycoumarin (7) (Inuma et al., 1993).

The IR spectrum of compound 8 showed a hydroxyl signal at 3368 cm⁻¹, a conjugated carbonyl ester absorbance at 1647 cm⁻¹ and aromatic C = C stretching absorptions at 1593 and 1466 cm⁻¹. The ¹H NMR spectrum showed characteristic signals of a flavonoid compound. An ABX pattern in the B-ring was revealed by the three protons resonances at δ 7.74 (1H, dd, J = 8.2 and 1.7 Hz), δ 7.76 (1H, J = 1.7 Hz) and δ 7.02 (1H, d, J = 8.2 Hz), which could be assigned to H-6´, H-2´ and H-5´, respectively, as found in 3′,4′-oxygenated flavonoids (Chen et al., 1997). Five methoxyl signals were observed at δ 3.84-4.07, suggesting that compound 8 was a polymethoxyflavone. The proton signal at δ 6.20 was suggestive of a phenolic hydroxyl at C-4′ and the low-field singlet at δ 12.36 is characteristic of the chelated 5-OH.

The ¹³C NMR spectrum of 8 showed four aromatic methoxyls, with considerable downfield chemical shifts at δ 60.1-62.0, that suggest these groups are attached to di-ortho-substituted carbons (Roitman & James, 1985) and possibly located on the A-ring and C-ring. The resonance at δ 56.0 could be assigned to the methoxyl group located at C-3′, considering this chemical shift is characteristic of an aromatic methoxyl attached to carbon bearing one or no ortho substituent.

The unambiguous assignments of carbons and hydrogens of compound 8 was possible based on the analysis of the HMBC cross correlations. The attribution of chemical shifts in rings A and C were made according to the following correlations: OH-5 with C-5 (δ 149.1) (²J), C-6 (δ 136.2) (³J) and C-10 (107.4) (³J); CH₃O-6 (δ 3.92) with C-6 (δ 136.2) (³J), CH₃O-7 (δ 4.07) with C-7 (δ 152.9) (³J), CH₃O-8 (δ 3.93) with C-8 (δ 132.8) (³J) and CH₃O-3 (δ 3.84) with C-3 (δ 138.6) (³J). Ring B was assigned by the interactions between H-2′ (δ 7.76) with C-2 (δ 155.9) (³J), C-4′ (δ 148.7) (³J) and C-6′ (δ 122.9) (³J); H-5′ (δ 7.02) with C-1′ (δ 122.5) (³J) and C-3′ (δ 146.5) (³J) and H-6′ (δ 7.74) with C-2 (δ 155.9) (³J), C-4′ (δ 148.7) (³J) and C-2′ (δ 110.9) (³J). All the correlations are shown in Figure 2. On the basis of the above evidence, compound 8 was identified as 5,4′-dihydroxy,3,6,7,8-3′-pentamethoxyflavone. Its chemical shifts were also compared with literature data (Sharaf et al., 1992).

Figure 2.  HMBC correlations of compound 8.

Table 1 . ¹H and ¹³C NMR chemical shifts assignments for compound 8.

Atom position	δH	δC
2		155.9
3		138.6
4		179.2
5		149.1
6		136.2
7		152.9
8		132.8
9		144.8
10		107.4
1′		122.5
2′	7.76 (s)	110.9
3′		146.5
4′		148.7
5′	7.02 (d, J = 8.2 Hz)	114.8
6′	7.74 (dd, J = 8.2, 1.7 Hz)	122.9
3	3.84 (s)	60.1
6	3.92 (s)	61.1
7	4.07 (s)	61.7
8	3.93 (s)	62.0
3′	3.94 (s)	56.0
OH-5	12.36 (s)

The ¹H NMR spectrum of compound 9 showed a group of signals for kaempferol and a sugar unit. Kaempferol was indicated by two doublets at δ 6.38 and δ 6.70 (J = 2.2 Hz) for H-6 and H-8 respectively and a pair of doublets at δ 8.06 and δ 6.88 (J = 8.9 Hz) for H-2′,6′ and H-3′,5′, respectively, referring to an AA′,BB′ system. The sugar region of the ¹H NMR spectrum revealed a doublet at δ 5.24 (J = 1.6 Hz) related to the anomeric proton H-1′′ of rhamnose. The other sugar protons resonated at δ 4.02 (dd, J = 3.4, 1.6 Hz, H-2′′), 3.83 (dd, J = 9.2, 3.4 Hz, H-3′′), 3.43 (t, J = 9.4 Hz, H-4′′) and their unambiguous assignments were further confirmed with the help of COSY and NOESY experiments. The HMBC spectrum proved that the rhamnosyl unit was attached at C-7 due to the correlation between the anomeric proton at δ 5.24 and the carbon at δ 163.2 (C-7) (³J). Supporting this information were the interactions between H-6 (δ 6.38) and H-8 (δ 6.70) with C-7 (²J). Thus, the structure of 9 was assigned as kaempferol 7-O-α-l-rhamnopyranoside (Kaouadji, 1990).

4′,5-Dihydroxy-3,6,7,8,3′-pentamethoxyflavone showed no antibacterial activity at 256 μg/mL against any strains of S. aureus used (MIC ≥ 512 μg/mL). When the compound was incorporated into the growing medium at 128 μg/mL (≤ ¼ MIC), a two-fold reduction in the MIC was observed for tetracycline and for erythromycin and a dramatic reduction of the MIC for norfloxacin (Table 2).

Table 2.  MIC values (μg/mL) of test strains for antibiotics in the absence and (presence) of 4′,5-dihydroxy-3,6,7,8,3′-pentamethoxyflavone (128 μg/mL).

Strain (efflux protein)	Norfloxacin	Tetracycline	Erythromycin
SA-1199B (NorA)	64 (2)	–	–
IS–58 (TetK)	–	32 (16)	–
RN4220 (MsrA)	–	–	64 (32)

4′,5-Dihydroxy-3,6,7,8,3′-pentamethoxyflavone modulated the activities of the antibiotics by reducing the concentration of antibiotic needed to inhibit the growth of the drug resistant (effluxing) bacteria. This activity may be related to its lipophilicity, a common feature of several putative efflux pump inhibitors. This quality, as pointed out by Gibbons (2004), is likely to be important for its solubility in the bacterial membrane and binding to the efflux transporters before inhibition can occur.

Some methoxylated flavones that potentiate the activity of antimicrobial drugs have been described (Stermitz et al., 2002). However, as far as we know, neither a pentamethoxyflavone nor a methoxylated flavone from the Malvaceae had been previously evaluated. Thus, the results here presented indicate that H. tiubae (and broadly Malvaceae) could serve as a source of plant-derived natural products that modulate bacterial multidrug resistance, i.e., a source of potential adjuvant of antibiotics.

Compound 8 (4′,5-dihydroxy-3,6,7,8,3′-pentamethoxyflavone): Yellow needles (hexane-CHCl₃), IR ν^KBr_max cm⁻¹: 3368, 3004, 2940, 2836, 1282, 1050. ¹H NMR (CDCl₃): see Table 1. ¹³C NMR (CDCl₃): see Table 1. NMR data agree with literature (Sharaf et al., 1992).

Compound 9 (kaempferol 7-O-α-l-rhamnopyranoside): Yellow powder (MeOH). ¹H NMR (CDCl₃): δ 8.06 (2H, d, J = 8.9 Hz, H-2´/6´), 6.88 (2H, d, J = 8,9 Hz, H-3´/5´), 6.70 (1H, d, J = 2.2 Hz, H-8), 6.38 (1H, d, J = 2.2 Hz, H-6), 5.54 (1H, d, J = 1.6 Hz, H-1′′), 4.03 (1H, dd, J = 3.4, 2 Hz, H-2′′), 3.83 (dd, J = 9.2, 3.4 Hz, H-3′′), 3.60 (1H, dd, J = 9.4, 6 Hz, H-5′′), 3.43 (1H, t, J = 9.4 Hz, H-4′′), 1.25 (1H, d, J = 5.8 Hz, H-6′′). ¹³C NMR (CDCl₃): δ 177.4 (C-4), 163.2 (C-7), 162.3 (C-28), 160.7 (C-4´), 157.3 (C-9), 148.7 (C-2), 137.5 (C-3), 130.8 (C-2´/6´), 123.5 (C-1´), 116.3 (C-3´/5´), 106.1 (C-10), 99.9 (C-6), 99.8 (C-1´´), 95.32 (C-8), 73.6 (C-4′′), 72.0 (C-3′′), 71.7 (C-2′′), 71.2 (C-5´´), 18.0 (C-6´´). NMR chemical shifts agree with literature (Kaouadji, 1990).

Acknowledgements

J.P.S-J. and V.S.F-S. are very grateful to Dr. Simon Gibbons (University of London) for his valuable and kind cooperation. CNPq (PIBIC/UFPb) is thanked for the award of an undergraduate studentship. This work was supported by the following Brazilian agencies: CNPq and CAPES.

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.