Profiling polyphenol composition by HPLC-DAD-ESI/MSn and the antibacterial activity of infusion preparations obtained from four medicinal plants

Abstract

The infusions of Thymus pallescens Noë, Saccocalyx satureioides Coss. et Dur., Ptychotis verticillata Briq. and Limoniastrum guyonianum Boiss. have been used as medicinal remedies for many diseases in Algerian folk medicine. These species have also been well documented as rich sources of phytochemicals, such as phenolic compounds with wide diversified chemical structures, which exhibit far-ranging biological activities. Thus, the phenolic compound profile of the aqueous extracts, obtained by infusing, of the mentioned species was obtained by HPLC-DAD-ESI/MS, and their antibacterial activity was evaluated against clinical isolates. Several phenolic acids were identified and quantified, particularly caffeic acid derivatives along with glycosylated flavonoids. T. pallescens and S. satureioides contain 13 phenolic compounds, where rosmarinic acid was the most abundant phenolic acid present, while L. guyonianum presented myricetin-3-O-glucoside and myricetin-O-rhamnoside as the main compounds among the eight detected molecules. P. verticillata presented a profile of ten phenolic compounds, where 5-O-caffeoylquinic acid was the most abundant phenolic acid, followed by the flavone luteolin-3-O-glucoside. The antibacterial activity of the infusions ranged between 2.5 and 20 mg mL⁻¹ (MIC values), and L. guyonianum showed the highest activity against all of the tested bacteria, Staphylococcus aureus and Pseudomonas aeruginosa being the most sensitive and resistant strains, respectively. Thus, the studied plant species are sources of natural antibacterial substances that can be used to fight against pathogenic microorganisms.

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1 Introduction

Phenolic compounds are generally part of a complex mixture isolated from plants and other matrices of biological origin.^1,2 They are interesting candidates for the discovery of new antimicrobial agents to be loaded into or coated onto biomaterials.^3,4 Consequently, it is essential to identify and measure all of the bioactive constituents of medicinal plants in order to ensure the reliability and repeatability of biological research as well as to enhance the quality control over the pharmacological benefits and/or hazards.^5,6 Liquid chromatography coupled with different detectors, such as a diode array detector (DAD) and a mass spectrometer (MS), plays a prominent role as an analytical tool for detecting and identifying active and/or reactive metabolites.^7,8 MS detection not only allows one to determine natural compounds’ chemical structures, but also offers excellent sensitivity with sufficient precision and selectivity within a reasonable time, playing an important role in the analysis of phenolic compounds (e.g. flavonoids, phenolic acids and others).^7,9–11

Antibiotic resistance is a serious and growing phenomenon in contemporary medicine and has emerged as one of the pre-eminent public health concerns in the 21st century.^4,12 Most pathogenic bacteria have developed resistance to modern antibiotics, as a result of which we are observing multi-drug resistance among bacterial strains.^4,13 Herbal medicine has been assumed to be an effective alternative to resolve this problem due to the bioactivities of herbs and many studies have been conducted across the globe to prove the antimicrobial efficiency and/or properties of several plants.^3,14,15

Algerian medicinal plants contain a great number of bioactive compounds with therapeutic effects,^16–18 and have been used since ancient times as herbal remedies.^19,20 However, the phytochemical constituents of many of these species are still unknown.^16,21Thymus pallescens Noë, Saccocalyx satureioides Coss. et Dur., Ptychotis verticillata Briq and Limoniastrum guyonianum Boiss. are some examples of unexplored medicinal plants from Algeria. T. pallescens (Lamiaceae family) is an endemic plant to the northern region of Algeria, widely used for its antitussive, antiseptic, expectorant, anti-helmintic and antispasmodic properties.²² While, S. satureioides, also from the Lamiaceae family, is an aromatic shrub (20–100 cm). This endemic plant is normally grown in septentrional Sahara, Algeria.^23,24 In folk medicine, the aerial parts are commonly used in water preparations (infusions and decoctions) for the treatments of gastric disorders and spasms,²⁵ as well as for diabetes.²⁶ The antimicrobial properties of its essential oils have been previously studied,^25,27,28 nonetheless, the polar extracts have been less explored. Ptychotis verticillata Briq (Apiaceae family), also known as Ammoides verticillata Briq or Ptychotis ammoides Koch. and commonly named Nûnkha,^29,30 is an aromatic herbaceous species (10–35 cm tall), endemic to northwestern Algeria and used for its culinary and medicinal properties as a febrifuge, antispasmodic, antiseptic and antidiabetic, especially in decoctions and infusions.^31,32 Moreover, the infusions and ethanolic extracts have been reported to have a wide diversity of polyphenols (flavonoids, saponins, and tannins), while its essential oils are mainly characterized by the presence of thymol and carvacrol.³³L. guyonianum (Plumbaginaceae family) is a medicinal halophyte species endemic to the north of Africa; the infusion of its leaves and galls are traditionally used as an anti-dysenteric against infectious diseases or parasites responsible for painful and bloody diarrhea.³⁴ Recent studies performed by Ziani et al.¹⁶ and Krifa et al.³⁵ demonstrated the antioxidant and antitumor potential of this plant.

To the best of the authors’ knowledge, the phytochemical analysis of T. pallescens, S. satureioides and P. verticillata has not been previously reported. The chemical characterization and simultaneous analysis of the most prominent compounds are therefore necessary to clearly understand the biological properties of these plants. The infusion preparations of these four medicinal plant species were chosen to continue a previous study of the authors,¹⁶ where their antioxidant and cytotoxic properties were reported and different hydrophilic bioactive compounds were also quantified spectrophotometrically. Therefore, the aim of the present work was to identify those bioactive compounds by using HPLC-DAD-ESI/MSn. Furthermore, the antibacterial activity of the infusions was also tested against six Gram-negative and four Gram-positive multi-resistant bacterial strains.

2 Materials and methods

2.1. Plant material

The aerial parts of the four species (Thymus pallescens Noë, Saccocalyx satureioides Coss. et Dur., Limoniastrum guyonianum Boiss. and Ptychotis verticillata Briq) were harvested from some semi-arid and arid areas in Algeria between April and May 2014 as described in a previous study,¹⁶ taking into account local consumers’ criteria for the seasoning use of these species and the optimal growth stage and gathering period. A voucher specimen of each species was deposited in the herbarium of the Department of Botany of the National Superior School of Agronomy (ENSA), where the taxonomic criteria of the authors^30,36 were used for botanical identification. After 40 days of shade-drying in the dark, all of the samples were ground for all of the subsequent analyses.

For the infusion preparation, 1 g of the plant material was added to 200 mL of boiling distilled water and left to infuse at room temperature for 5 min, and then filtered through Whatman paper. The obtained infusions were frozen at −20 °C and lyophilized for further analyses.

2.2. Standards and reagents

Acetonitrile and methanol HPLC-grade and formic acids were purchased from Fisher Scientific (Lisbon, Portugal). Phenolic compound standards were obtained from Extrasynthese (Genay, France) and their purities were above 97%, as determined by HPLC-DAD analysis and stock solutions of these compounds (1 mg mL⁻¹) were prepared in methanol/water (20 : 80, v/v). The culture media Mueller–Hinton broth (MHB) and tryptic soy broth (TSB) as well as blood agar with 7% sheep blood and MacConkey agar plates were obtained from Biomerieux (Marcyl'Etoile, France). The dye p-iodonitrotetrazolium chloride (INT) was purchased from Sigma-Aldrich (St Louis, MO, USA) and was used as a microbial growth indicator. Water was treated using the Milli-Q water purification system (TGI Pure WaterSystems, Greenville, SC, USA).

2.3. Phenolic compound characterization by HPLC-DAD-ESI/MSn

For the LC/MS analysis, lyophilized infusion extracts were dissolved in water at a concentration of 10 mg mL⁻¹ and filtered through a 0.22 μm disposable LC filter disk prior to the analysis. Spectral UV-Vis data from all peaks were collected in the range of 240–600 nm, and chromatograms were recorded at 370, 330 and 280 nm for phenolic compound analysis. The HPLC-DAD-ESI/MSn (Dionex Ultimate 3000 UPLC) consisted of a diode array detector (DAD) connected to a Linear Ion Trap LTQ XL mass spectrometer (Thermo Scientific, San Jose, CA, USA), following a procedure previously reported by the authors.³⁷ Chromatographic separation was performed using a Waters Spherisorb S3ODS-2 C18 automated column (3 μm, 4.6 × 150 mm, Waters, Milford, MA, USA) and the mass detection was performed using ESI, operating in negative mode. An Xcalibur®data system (ThermoFinnigan, San Jose, CA, USA) was used for data acquisition.

Phenolic compound identification was achieved by comparing retention times and UV–vis and mass spectra with those of available standard compounds. Otherwise, the available data reported in the literature were applied to identify the compounds. The quantification of these compounds was calculated from the calibration curves (2.5–100 μg mL⁻¹) of each available phenolic standard or by using the most similar standard available. The results were expressed as mean values ± standard deviations (SD), in mg g⁻¹ of lyophilized infusion extract.

2.4. Evaluation of the antibacterial activity

2.4.1. Microorganisms and culture conditions. The tested bacterial strains were clinical isolates obtained from patients hospitalized in the Local Health Unit of Bragança and Hospital Centre of Trás-os-Montes and Alto-Douro, Vila Real, northeast of Portugal. Six Gram-negative bacteria: Morganella morganii and Pseudomonas aeruginosa isolated from expectoration, Escherichia coli, extended spectrum beta-lactamase (ESBL)-producing E. coli, Klebsiella pneumoniae and extended-spectrum beta-lactamase (ESBL)-producing K. pneumoniae isolated from urine; and four Gram-positive bacteria: Enterococcus faecalis isolated from urine, Listeria monocytogenes isolated from cerebrospinal fluid, methicillin-sensitive Staphylococcus aureus (MSSA) isolated from wound exudate, and methicillin-resistant Staphylococcus aureus (MRSA) isolated from expectoration were tested. All strains were identified and characterized regarding the antibiotic susceptibility using the MicroScan® panels automated methodology (Siemens, Medical Solutions Diagnostics Camberley, West Sacramento, CA, USA). The interpretation criteria were based on interpretive breakpoints as indicated in Clinical and Laboratory Standards Institute,³⁸ and in the European Committee on Antimicrobial Susceptibility Testing.³⁹ Table S1 (ESI†) shows the sensitivity/resistance profiles of the different antibiotics tested for each clinical isolate.

2.4.2. Determination of the minimal inhibitory concentrations (MICs). A microdilution method and the rapid p-iodonitrotetrazolium chloride (INT) colorimetric assay were used to determine the minimum inhibitory concentration (MIC).⁴⁰

Briefly, stock solutions of 100 mg mL⁻¹ were prepared and 100 μL of each stock solution (100 mg mL⁻¹) was diluted in 400 μL of MHB or TSB media according to bacteria requirements (making a solution of 20 mg mL⁻¹). Then, 200 μL of this extract solution was then added to the first well of a microplate (96-well microplate) and 100 μL from the first well were pipetted to other wells containing 100 μL of media making successive dilutions.

Afterwards, 10 μL of inoculum (1.5 × 10⁸ CFU mL⁻¹) of fresh overnight cultures of bacteria was added to all of the wells containing the test concentrations in the range of 20 to 0.156 mg mL⁻¹. Three negative controls were prepared (one with MHB/TSB, another one with the extract, and the third one with the medium, inoculum and antibiotic).

The MIC of the samples was determined after adding INT (0.2 mg mL⁻¹, 40 μL) and after incubation at 37 °C in an oven (Jouan, Berlin, Germany) for 30 min where the viable microorganisms reduced the yellow dye to pink. The MIC was defined as the lowest extract concentration that prevented this change and exhibited the complete inhibition of bacterial growth.

2.5. Statistical analysis

Three repetitions of each sample and triplicates of the infusion preparations were used in each assay. In order to determine the significant difference among samples, a Student's t-test (with p = 0.05, significant differences between samples) was applied (IBM SPSS Statistics for Windows, version 23.0, IBM Corp., Armonk, New York, USA).

3 Results and discussion

3.1. HPLC-DAD-ESI/MSn analysis of phenolic compounds

The HPLC-DAD-ESI/MSn chromatographic profiles of the studied plants are shown in Fig. 1 and 2. Tables 1–3 list all of the characterized metabolites obtained by the chromatographic analysis.

Fig. 1 HPLC-DAD-ESI/MSn phenolic profile of Thymus pallescens (A) and Saccocalyx satureioides (B) infusions, recorded at 280 nm.

Fig. 2 HPLC-DAD-ESI/MSn phenolic profile of Limoniastrum guyonianum (A) and Ptychotis verticillata (B) infusions, recorded at 370 nm.

Table 1 Retention time (R_t), wavelengths of maximum absorption in the visible region (λ_max), mass spectral data, tentative identification and quantification of phenolic compounds in Thymus pallescens and Saccocalyx satureioides infusions

Peak	R t (min)	λ max (nm)	[M − H]^− (m/z)	MS^2 (m/z)	Tentative identification	Quantification (mg g^−1 extract)		t-Student's test p-value
						Thymus pallescens	Saccocalyx satureioides
1	7.32	326	353	191(100), 179(5), 161(3), 135(3)	5-O-Caffeoylquinic acid	2.59 ± 0.04	nd	—
2	10.13	327	593	473(3), 383(15), 353(2), 311(3), 297(36)	Apigenin-6,8-C-dihexoside	2.54 ± 0.01	12.2 ± 0.4	<0.001
3	12.57	336	637	461(100), 285(7)	Luteolin-O-diglucuronide	nd	5.0 ± 0.2	—
4	14.55	320	521	359(100), 197(4), 179(4), 161(3), 135(2)	Rosmarinic acid hexoside	nd	1.68 ± 0.08	—
5	15.28	342	477	301(100)	Quercetin-3-O-glucoside	2.17 ± 0.07	nd	—
6	16.1	334	637	461(100), 285(20)	Luteolin-O-diglucuronide	1.23 ± 0.01	nd	—
7	17.94	343	593	285(100)	Luteolin-7-O-rutinoside	1.30 ± 0.01	1.22 ± 0.01	<0.001
8	18.58	347	461	285(100)	Luteolin-7-O-glucuronide	11.5 ± 0.3	5.2 ± 0.2	<0.001
9	19.53	283, 328	719	539(25), 521(20), 359(100), 297(3), 179(36), 161(2), 135(3)	Sagerinic acid	2.78 ± 0.07	2.39 ± 0.01	<0.001
10	20.5	334	549	505(30), 463(100), 301(20)	Quercetin-O-malonyhexoside	nd	1.11 ± 0.01	—
11	21.71	288, 323	555	537(5), 511(8), 493(10), 311(100), 197(19), 179(3), 161(3), 135(12)	Salvianolic acid K	5.72 ± 0.01	2.72 ± 0.02	<0.001
12	22.24	328	359	197(27), 179(37), 161(100), 135(2)	Rosmarinic acid	43.1 ± 0.8	50.1 ± 0.4	<0.001
13	23.11	335	445	269(100)	Apigenin-O-glucuronide	4.56 ± 0.05	3.3 ± 0.1	<0.001
14	24.74	340	461	285(100)	Kaempferol-O-glucuronide	29.1 ± 0.5	1.35 ± 0.03	<0.001
15	25.54	329	537	493(8), 359(100), 313(11), 295(4), 197(2), 179(3), 161(2)	Lithospermic acid A isomer I	20.9 ± 0.9	21.69 ± 0.02	0.107
16	30.76	329	537	493(9), 359(100), 313(9), 295(3), 197(3), 179(3), 161(4)	Lithospermic acid A isomer II	2.7 ± 0.1	2.22 ± 0.03	<0.001
					Total phenolic acid	78 ± 2	80.8 ± 0.5	0.020
					Total flavonoids	52.4 ± 0.9	29.3 ± 0.9	<0.001
					Total phenolic compounds	130 ± 3	110 ± 1	<0.001

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Table 2 Retention time (R_t), wavelengths of maximum absorption in the visible region (λ_max), mass spectral data, tentative identification and quantification of phenolic compounds in Limoniastrum guyonianum infusion

Peak	R t (min)	λ max (nm)	[M − H]^− (m/z)	MS^2 (m/z)	Tentative identification	Quantification (mg g^−1 extract)
1^Lg	13.99	353	631	479(5), 317(100)	Myricetin-hexosyl-gallate	5.73 ± 0.05
2^Lg	14.61	353	493	317(100)	Myricetin-3-O-glucuronide	6.90 ± 0.08
3^Lg	15.21	356	479	317(100)	Myricetin-3-O-glucoside	8.2 ± 0.2
4^Lg	17.5	354	535	317(100)	Myricetin-O-acetylglucoronide	5.41 ± 0.02
5^Lg	17.83	349	463	317(100)	Myricetin-3-O-rhamnoside	8.12 ± 0.05
6^Lg	19.75	355	535	317(100)	Myricetin-O-acetylglucoronide	5.36 ± 0.06
7^Lg	20.72	352	659	493(12), 479(100), 317(51)	Myricetin derivative	5.17 ± 0.03
8^Lg	22.33	353	535	317(100)	Myricetin-O-acetylglucoronide	5.36 ± 0.01
					Total phenolic compounds	50.3 ± 0.5

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Table 3 Retention time (R_t), wavelengths of maximum absorption in the visible region (λ_max), mass spectral data, tentative identification and quantification of phenolic compounds in Ptychotis verticillata infusion

Peak	R t (min)	λ max (nm)	[M − H]^− (m/z)	MS^2 (m/z)	Tentative identification	Quantification (mg g^−1 extract)
1^Pv	4.94	323	353	191(100), 179(45), 161(3), 135(8)	3-O-Caffeoylquinic acid	3.04 ± 0.10
2^Pv	6.77	325	353	191(11), 179(2), 173(100), 161(1), 135(4)	4-O-Caffeoylquinic acid	3.42 ± 0.15
3^Pv	7.31	326	353	191(100), 179(2), 161(3), 135(4)	5-O-Caffeoylquinic acid	40.90 ± 1.32
4^Pv	18.28	347	593	285(100)	Luteolin-7-O-rutinoside	1.49 ± 0.03
5^Pv	19.21	347	447	285(100)	Luteolin-7-O-glucoside	5.99 ± 0.22
6^Pv	19.93	347	609	285(100)	Kaempferol-O-dihexoside	0.75 ± 0.03
7^Pv	20.42	347	505	463(25), 301(100)	Quercetin-O-acetylhexoside	1.20 ± 0.03
8^Pv	23.46	332	431	269(100)	Apigenin-7-O-glucoside	2.67 ± 0.03
9^Pv	23.79	347	447	285(100)	Kaempferol-O-glucuronide	5.44 ± 0.32
10^Pv	24.2	337	489	285(100)	Kaempferol-O-acetylhexoside	0.74 ± 0.01
					Total phenolic acid	47.35 ± 1.57
					Total flavonoids	18.28 ± 0.67
					Total phenolic compounds	65.63 ± 2.25

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Regarding the phenolic composition of Thymus pallescens and Saccocalyx satureioides, as both species belong to the Lamiaceae family, the profiles were very similar to other species belonging to this family.^41–43 Thirteen compounds (six phenolic acid derivatives and seven flavonoids) were identified in both species, based on their chromatographic behavior and mass spectra, in comparison with the literature. Table 1 summarizes the identified compounds, their retention times and m/z values for the parent ion and fragment ions. Both samples presented flavonol derivatives (quercetin, kaempferol glycoside derivatives), flavone derivatives (apigenin and luteolin glycoside derivatives) and phenolic acid derivatives, mainly caffeic acid derivatives, such as caffeic acid dimers, trimers, and tetramers (dimers of rosmarinic acid). To the best of the authors’ knowledge, this is the first report regarding the phenolic composition of these two species.

Compounds 1, 5, 7, 8 and 12 were identified as 5-O-caffeoylquinic acid, quercetin-3-O-glucoside, luteolin-7-O-rutinoside, luteolin-7-O-glucuronide and rosmarinic acid, respectively, according to their DAD spectra, mass characteristics and retention times when compared with commercial standards. With the exception of compounds 1 and 5, which were only present in T. pallescens and compound 4 that was only present in S. satureioides, the other compounds were found in both samples.

Compounds 4, 9, 11, 13, 15 and 16 were identified as caffeic acid derivatives. Compound 4 ([M − H]⁻ at m/z 521) fragmented at m/z 359 (rosmarinic acid, loss of −162 mu, hexoside moiety), which allowed its identification as rosmarinic acid hexoside. Compound 9 ([M − H]⁻ at m/z 719) released a main MS² fragment at m/z 359 ([M − 2H]²⁻, rosmarinic acid), allowing its identification as sagerinic acid.⁴⁴ Compounds 15 and 16 ([M − H]⁻ at m/z 537) revealed a similar UV spectrum and fragmentation pattern, being identified as a caffeic acid trimer, lithospermic acid A, taking into account literature findings.^44–47 Compound 11 ([M − H]⁻ at m/z 555) was tentatively assigned to salvianolic acid K, due to a similar fragmentation pattern described by Hauck et al.⁴⁸

The remaining molecules (peaks 2, 3, 6, 10, 13 and 14) correspond to flavonoid derivatives. Compound 2 ([M − H]⁻ at m/z 593) released the MS² fragment ions at m/z 473 and 383 (loss of 120 and 90 mu characteristic of C-hexosyl flavones) and at m/z 353 corresponding to apigenin aglycone (apigenin + 83 mu, bearing some sugar residues),⁴⁹ which allowed the identification of this compound as apigenin-C-hexoside C-hexoside. Thus, this compound was tentatively identified as apigenin-6,8-C-diglucoside. Compounds 3 and 6 presented a UV spectrum characteristic of luteolin (λ_max at 350 nm) and the same pseudomolecular ion [M − H]⁻ at m/z 637, releasing two fragments at m/z 461 and 285 ([M − 176 − 176]⁻, loss of two glucuronyl moieties), being identified as luteolin-O-diglucuronide. Similar findings were taken into account to identify compounds 13 and 14, which were assigned to apigenin-O-glucuronide and kaemferol-O-glucuronide, respectively. Compound 3 was only detected in S. satureioides, while compound 6 was identified in T. pallescens. Finally, compound 10 ([M − H]⁻ at m/z 549), releasing MS² fragments at m/z 301 ([M − H − 162 − 86]⁻, loss of a malonylhexoside moiety) was assigned to quercetin-O-malonylhexoside, being only present in S. satureioides. Rosmarinic acid was the most abundant phenolic acid present in both samples, while luteolin-7-O-glucuronide was the most abundant flavonoid present in T. pallescens and apigenin-6,8-C-dihexoside was predominant in S. satureioides.

In the case of L. guyonianum infusion, only flavonol glycoside derivatives were detected, related to myricetin (λ_max around 354 nm, an MS² fragment at m/z 317). They presented MS² fragments corresponding to distinct losses of glucuronyl (−176 mu), glucosyl (−162 mu) and rhamnosyl (−146 mu) moieties, and an elution order coherent with the type of substituent sugar, according to their expected polarity, being assigned to myricetin-O-glucuronide (peak 2^Lg), myricetin-3-O-glucoside (peak 3^Lg, positively identified with the commercial standards), and myricetin-O-rhamnoside (peak 5^Lg), respectively. Myricetin and its derivatives have been previously described in Limoniastrum feei (Girard) Batt from Algeria⁵⁰ as myricetin-3-O-beta-galactopyranoside and myricetin-3-O-alpha-rhamnopyranoside. Taking into account the position and nature of the sugar moieties, peaks 2^Lg and 5^Lg were assumed to be myricetin-3-O-glucuronide and myricetin-3-O-rhamnoside, respectively.

Furthermore, compound 1^Lg was identified as a hexosyl derivative of myricetin that also appeared to be attached to a gallic acid moiety (−152 mu), yielding a deprotonated ion at m/z 631, which consisted of a myricetin-hexosyl-gallate. Compounds 4^Lg, 6^Lg and 8^Lg presented the same pseudomolecular ion [M − H]⁻ at m/z 505, releasing an MS² fragment at m/z 317 (myricetin; [M − H − 42 − 162]⁻, loss of an acetylhexoside moiety), being assigned to myricetin-O-acetylhexoside. No further identification was possible to obtain for peak 7^Lg ([M − H]⁻ at m/z 659), being assigned to a myricetin derivative. The major myricetin derivatives present in this sample were myricetin-3-O-glucoside and myricetin-O-rhamnoside, respectively.

The phenolic profile of P. verticillata infusion, determined by HPLC-DAD-ESI/MSn, presented three chlorogenic acids, being compounds 1, 2 and 3 ([M − H]⁻ at m/z 353) identified as 3-O-caffeoylquinic acid, 4-O-caffeoylquinic acid and 5-O-caffeoylquinic acid, respectively, according to their similar fragmentation pattern previously described by Clifford et al.^51,52 The remaining compounds were identified as flavonols (peaks 6^Pv, 7^Pv, 9^Pv and 10^Pv) and flavones (4^Pv, 5^Pv and 8^Pv). The latter compounds 4^Pv (luteolin-3-O-rutinoside), 5^Pv (luteolin-3-O-glucuronide) and 8^Pv (apigenin-3-O-glucoside) were positively identified with commercial standards. Peak 7^Pv ([M − H]⁻ at m/z 505) released two fragments, at m/z 463 (−42, loss of a acetyl moiety) and m/z 301 (−162 u, loss of a hexosyl moiety), thus being tentatively assigned to quercetin-O-acetylhexoside. Compounds 6^Pv ([M − H]⁻ at m/z 609), 9^Pv ([M − H]⁻ at m/z 447) and 10^Pv ([M − H]⁻ at m/z 489) were identified as kaempferol glycosides based on their UV spectra (λ_max around 348 nm) and the production of an MS² fragment ion at m/z 285, and could be assumed as kaempferol-O-dihexoside, kaempferol-O-glucuronide and kaempferol-O-acetylhexoside, respectively. 5-O-Caffeoylquinic acid was the most abundant compound, followed by the flavone luteolin-3-O-glucoside.

3.2. Antibacterial activity

The search for natural antimicrobial compounds in clinical microbiology is incited by the need to thwart the increasing infectious diseases caused by multiple drug resistant (MDR) and total drug resistant (TDR) strains.⁴ In the biomedical field, the microbial antibiotic-resistance leads to a growing need for new, effective anti-infective materials for the prevention and delay of implant and device-associated infections.⁵³ In the present work, the antibacterial activity of the samples was tested against ten bacteria, some of them being multiresistant. The infusion of L. guyonianum showed the highest activity against E. coli, E. coli ESBL, MRSA and MSSA (MICs = 2.5 mg mL⁻¹), followed by K. pneumoniae, K. pneumoniae ESBL, E. faecalis, L. monocytogenes (MICs = 5 mg mL⁻¹), and P. aeruginosa and M. morganii (MICs ranging between 10–20 mg mL⁻¹). The infusions of T. pallescens and S. satureioides showed almost similar values and were found to be less active against K. pneumoniae, K. pneumoniae ESBL, E. faecalis, L. monocytogenes, P. aeruginosa and M. morganii strains (MICs ranging from 2.5 to >20 mg mL⁻¹) (Table 4). P. verticillata infusion showed the weakest activity against all of the bacteria presenting MIC values in the highest tested concentration or even presenting no activity at the maximum tested concentration: 20 mg mL⁻¹. These results showed that the plant infusions can inhibit bacterial strains irrespective of their mechanisms of resistance. No significant differences were observed between the strains showing well-known mechanisms of resistance and the susceptible ones, because one-dilution differences in this kind of analysis are taken for granted.

Table 4 MIC values (expressed in mg mL⁻¹) of Thymus pallescens, Saccocalyx satureioides, Limoniastrum guyonianum and Ptychotis verticillata infusions against six Gram-negative and four Gram-positive bacterial strains

Antimicrobial activity MIC values (mg mL^−1)	Thymus pallescens	Saccocalyx satureioides	Limoniastrum guyonianum	Ptychotis verticillata
MRSA, methicillin-resistant Staphylococcus aureus; MSSA, methicillin-susceptible Staphylococcus aureus; MIC, minimal inhibitory concentration; ESBL, extended-spectrum beta-lactamases.
Gram-negative bacteria
Escherichia coli	10	5	2.5	20
Escherichia coli ESBL	10	5	2.5	20
Klebsiella pneumoniae	10	10	5	20
Klebsiella pneumoniae ESBL	10	10	5	20
Morganella morganii	20	>20	20	>20
Pseudomonas aeruginosa	20	20	10	>20
Gram-positive bacteria
Enterococcus faecalis	10	10	5	20
Listeria monocytogenes	10	10	5	20
MRSA	5	5	2.5	10
MSSA	5	5	2.5	10

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Many research groups have gone one step further and isolated and identified the structure of flavonoids that possess antibacterial activity. The studied infusions were found to be rich sources of phenolic components that could exert antibacterial activity. For instance, flavonols and their derivatives, as they were found abundantly in the infusion of T. pallescens (quercetin and kaempferol) and L. guyonianum (myricetin), are characterized by a remarkable antibacterial activity against both Gram-positive and Gram-negative bacteria, such as S. aureus, Lactobacillus acidophilus, Porphyromonas gingivalis, Prevotella melaninogenica, S. epidermidis, E. coli, Proteus vulgaris, P. aeruginosa, Enterococcus aerogenes and Enterobacter sakazakii.^42,54 Quercetin, 3-O-methylquercetin and several quercetin glycosides,^55–59 and also kaempferol and its glycoside derivatives,^55,57,60 were also reported to have antimicrobial activity. Other categories of compounds with known antibacterial activity are phenolic acids, such as chlorogenic, protocatechuic, p-coumaric, caffeic, syringic, p-hydroxybenzoic, ferulic, vanillic, gentisic and gallic acids,¹² their derivatives,⁴² and flavones (e.g. apigenin and luteolin glycoside derivatives),^{12,55,56,61–65} isoflavones,^66–69 flavanones,^56,70 and other flavonol glycosides.^56,71–74

To the best of the authors’ knowledge, no data are available on the antibacterial activity of these plants against drug-resistant bacteria in the literature. The obtained results showed that the antibacterial activity of all of the plant infusions is moderate, especially against Gram-positive bacteria. Moreover, whilst methicillin-resistant strains usually display resistance to several drugs, no relevant differences were observed between methicillin-susceptible and -resistant strains. These data are also in agreement with earlier studies carried out on other species belonging to the genus of the studied plants. Aqueous extracts obtained from Thymus vulgaris showed antibacterial activity against many ATCC bacterial strains.^42,75,76 Moreover, many solvent fractions of L. guyonianum from Tunisia showed antibacterial activity against several human pathogenic strains such as S. aureus, E. feacalis, E. coli, Salmonella typhi and P. aeruginosa;⁷⁷ also, the Tunisian L. guyonianum methanolic, chloroformic and petroleum extracts showed a potent action against P. aeruginosa and S. aureus with MIC values of 23 and 46 μg mL⁻¹ as the best values, respectively.⁷⁸ Concerning the antimicrobial activity of S. satureioides, only the essential oil was previously studied by the authors,²⁵ and showed strong potential against ATCC bacterial strains (Bacillus subtilis, S. aureus, L. monocytogenes, E. faecalis, E. coli, K. pneumoniae, Proteus mirabilis, P. aeruginosa and Salmonella typhimurium).

According to the results obtained from the HPLC-DAD-ESI/MSn analysis, the appreciable antibacterial activity of the extracts against MDR strains could be explained by the wide spectrum of polyphenols identified in the infusions (previous section), which act as antimicrobial substances via different mechanisms of action (MA). In fact, many MA are ascribed to polyphenols, such as cytoplasmic membrane damage, and inhibition of nucleic acid, cell wall and cell membrane synthesis.^3,55 Moreover, in addition to their direct antibacterial activity, a growing body of evidence suggests that polyphenols may interfere with some bacterial virulence factors such as enzymes, toxins and signal receptors.^3,55,56,73

4 Conclusion

Overall, this work focuses on the determination of the phenolic compound profile as well as on the investigation of the antibacterial activity of four medicinal plant species from the Algerian flora. The identification and quantification of phenolic compounds in T. pallescens, S. satureioides, L. guyonianum and P. verticillata have been accomplished. The results indicate that the infusion preparation of T. pallescens and S. satureioides contained considerable amounts of rosmarinic acid, lithospermic acid A, luteolin-7-O-glucuronide and apigenin-6,8-C-dihexoside, while L. guyonianum was rich in myricetin derivatives (glucosides) and P. verticillata was rich in caffeic acid derivatives particularly 5-O-caffeoylquinic acid. The findings of this study showed that all four species exhibited a broad spectrum of antibacterial activity against clinical isolates (MIC values between 2.5 and 20 mg mL⁻¹), which could be used as an alternative source of antibiotics. However, pharmacological testing is necessary following the isolation of the bioactive compounds. The infusions of these plants should be furthermore investigated in vivo to better understand their efficacy and medicinal properties.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

The authors are grateful to the Foundation for Science and Technology (FCT, Portugal) and FEDER under Programme PT2020 for financial support to CIMO (UID/AGR/00690/2013), S. Heleno (SFRH/BPD/101413/2014) grant and L. Barros contract. The authors are also grateful to the Interreg España-Portugal for financial support through the project 0377_Iberphenol_6_E.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c7fo01315a

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