Next Article in Journal
Organic Matter Preservation in Ancient Soils of Earth and Mars
Previous Article in Journal
Embelin as Lead Compound for New Neuroserpin Polymerization Inhibitors
Previous Article in Special Issue
Almond Skin Extracts and Chlorogenic Acid Delay Chronological Aging and Enhanced Oxidative Stress Response in Yeast
Article

UPLC-MS/MS Phytochemical Analysis of Two Croatian Cistus Species and Their Biological Activity

1
Faculty of Chemistry and Technology, University of Split, 21000 Split, Croatia
2
Faculty of Science, University of Split, 21000 Split, Croatia
3
School of Medicine, University of Split, 21000 Split, Croatia
4
Faculty of Food Technology and Biotechnology, University of Zagreb, 10000 Zagreb, Croatia
*
Author to whom correspondence should be addressed.
Life 2020, 10(7), 112; https://doi.org/10.3390/life10070112
Received: 29 May 2020 / Revised: 6 July 2020 / Accepted: 9 July 2020 / Published: 14 July 2020

Abstract

Aqueous extracts of two Cistus species wild growing in Croatia—Cistus creticus (CC) and Cistus salviifolius (CS)—have been assessed with UPLC-MS/MS, showing 43 different phytochemicals, with flavonol glycosides: myricetin-3-hexoside and myricetin-rhamnoside, predominate ones in CC and myricetin-3-hexoside in CS. Antioxidant potential tested with the FRAP method showed no difference between CS and CC aqueous extracts, while higher phenolic content of CC comparing to CS, determined with a Folin–Cicolateu reagent correlated to its higher antioxidant capacity observed by the DPPH method. Both extracts were assessed for antimicrobial activity, using disc-diffusion and broth microdilution assays, targeting the opportunistic pathogens, associated with food poisoning, urinary, respiratory tract, blood stream and wound infections in humans. Antimicrobial assays revealed that fungi were in general more sensitive to both Cistus aqueous extracts, comparing to the bacteria where two extracts showed very similar activity. The most potent activity was observed against A. baumannii for both extracts. The extracts were tested on human lung cancer (A549) cell line using the MTT assay, showing very similar antiproliferative activity. After 72 h treatment with CC and CS aqueous extracts in concentration of 0.5 g/L, the viability of the cells were 37% and 50% respectively, compared to non-treated cells.
Keywords: antimicrobial; antioxidant; antiproliferative; Cistus; flavonoid; UPLC-MS/MS antimicrobial; antioxidant; antiproliferative; Cistus; flavonoid; UPLC-MS/MS

1. Introduction

The Cistus genus belongs to a small family of Cistaceae of eight genera, widely distributed in the Mediterranean. The species are known as rock roses and have noteworthy morphological diversification. This genus has several medicinal perennial shrubs widely distributed in the Mediterranean area [1].
Since ancient times, Cistaceae have been used in ethnomedicine due to their pharmacological potential against a broad range of disorders, including various skin diseases, diarrhea, ulcers, dysentery, catarrh, menstruation difficulties, and gout due to a number of natural pharmacological compounds they consist of [2,3,4]. According to ethno-pharmacology, the Cistus species has been used due to its antimicrobial, antiproliferative, anticancer, antiviral, antioxidant, anti-inflammatory, antiulcerogenic, antidiarrheal, and antispasmodic activity [5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26]. It is also known that some Cistus species have been used in human and animal diets, such as goats, lamb and beef [27,28,29,30,31,32,33,34].
Phytochemical profile of different Cistus species aqueous extracts reveals their polyphenolic profile, having flavonoids, phenolic acids, and ellagitanins as their main constituents [2,3,6,8,10,15,23,26,31,32,35,36,37,38,39,40,41,42,43,44].
This study examined two wild growing Croatian Cistus species, Cistus salviifolius (CS) and Cistus creticus (CC), known to be used in herbal tea preparation in human diet [34]. To the best of our knowledge this is the first UPLC-MS/MS phytochemical phenolic profile analysis of C. creticus and C. salvifolius aqueous extract. Due to the traditional use of Cistus species in human and animal diet, as well as for their pharmacological potential in ethno-medicine, we have examined the biological activity of CC and CS aqueous extracts. The pharmacological potential of two Cistus aqueous extracts was tested against the emerging opportunistic pathogens associated with skin, nail, gastrointestinal, bloodstream and respiratory infections, as well as their in vitro antiproliferative activity, antioxidant potential and total phenolic content.

2. Materials and Methods

2.1. Plant Material

Plant material, the upper part, stems, leaves, and flowers, of two species of the genus Cistus (rock rose)—Cistus salviifolius L. (sage-leaved rock rose), Cistus creticus L. (pink rock rose)—were collected on the island of Čiovo (Split, Croatia) in June 2015 and identified by a botanist, Jure Kamenjarin, Faculty of Science, University of Split. Voucher specimens of plant materials were deposited in the Department of Biochemistry, Faculty of Chemistry and Technology, Split, Croatia with numbers CS_2015_01 and CC_2015_01. Plant material was air-dried at room temperature for 3 days and 20 g of the dried material was chopped and extracted with 0.15 L of hot distilled water. Water solution was filtered after 30 min and water extracts were subjected to water evaporation by a low vacuum using the rotary evaporator. Dried aqueous extract was dissolved in distilled water and kept in a fridge at a temperature of −20 °C for further assays.

2.2. UPLC-MS/MS Phytochemical Analysis of Plant Extracts

Standard preparation for UPLC-MS/MS analysis was made as follows: stock standard solutions of each phenolic standard were prepared by dissolving in methanol at concentration of 0.1 g/L. Multi compound standard solutions were prepared in methanol, and dilutions from this solution were done in the range 0.00001–0.1 g/L for external calibration and validation experiments of the UPLC-MS/MS system. The three injections per level were done and peak area of each standard were used to make the respective standard curves. UPLC analyses of aqueous extracts were performed on Agilent 1290 RRLC instrument (Agilent Technologies, Santa Clara, CA, USA) coupled with binary gradient pump, autosampler, and column compartment. The gradient conditions were set up according to the methods reported [45]. The column used for separation was Zorbax Eclipse Plus C18 column (100 × 2.1 mm, 1.8 µm) (Agilent, Santa Clara, CA, USA).
Mass spectrometry experiments and optimization of the method were performed using a triple quadrupole mass spectrometer (QQQ 6430, Agilent, Santa Clara, CA, USA). The mass spectrometer was used in the dynamic multiple reaction monitoring mode (dMRM) in the ESI-positive and negative mode and operated with the following source parameters: capillary voltage, +4000/−3500 V, nitrogen drying gas temperature maintained at 300 °C with a flow rate of 11 L/h and the pressure of the nebulizer was set at 40 psi. Mass hunter software was used for data acquisition and analysis. Identification of the phenolic compounds was performed by comparing retention times and mass spectra with those of the authentic standards. In case of unavailability of standards, the prediction and structural identification of phenolic compounds was carried out by comparing the mass fragments with the previously reported mass fragmentation patterns [42,46]. Analytical method quality parameters, such as calibration curve, instrumental detection (LOD) and quantification (LOQ) limits were determined as previously reported by Zorić [45]. Quantitative data for all phenolic compounds were obtained by calibration curves of known standards. If a commercial standard was not available, quantification was performed using the calibration curve of standards from the same phenolic group. Results are shown as the mean values with the standard deviation.

2.3. DPPH Test

The antiradical activity of Cistus aqueous extracts was determined using the DPPH (2,2′-diphenyl-1-picrylhydrazy) radical scavenging method [47]. The antiradical activities of the samples were calculated according to the formula:
% inhibition = [(A0 − Asample)/A0] × 100,
where A0 was absorbance of the DPPH ethanolic solution measured at the beginning and Asample was absorbance of the sample measured after 60 min. The results were expressed as IC50 or the concentration of the extract that gives 50% of the inhibition of DPPH radical reaction.

2.4. FRAP Assay

The reducing antioxidant power of two Cistus aqueous extracts was determined using the FRAP method [48]. FRAP assay measures the ability of plant extracts to reduce the ferric 2,4,6-tripyridyl-s-triazine complex [Fe(III)–(TPTZ)2]2þ to the intensely blue colored ferrous complex [Fe(II)–(TPTZ)2]2þ in acidic medium. Reducing power of samples was calculated comparing the reaction signal given with solution of Fe2+ ions in known concentrations and expressed as mM equivalents of Fe2+ ions.

2.5. Total Phenolic Content

Total phenolic content was measured spectrophotometrically according to the Folin–Ciocalteu colorimetric method [49]. Gallic acid (Sigma-Aldrich, Steinheim, Germany) was used as a standard and samples were taken in triplicate, while the results were expressed as gallic acid equivalents.

2.6. MTT Assay

Lung cancer cell line A549 was grown at 37 °C in a humidified incubator and 5% CO2 in Dulbecco’s Modified Eagle Medium (DMEM, EuroClone, Milano, Italy) containing 10% fetal bovine serum and 1% antibiotics (Penicillin Streptomycin, EuroClone, Milan, Italy). Cell viability was determined using the tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide; MTT Sigma-Aldrich) in reduction MTT assay (Hussain, Nouri, and Oliver, 1993). An equal number of cells were seeded into the wells of a 96-well plate and allowed to attach overnight. Cells were afterward treated with both extracts (100 μL), in triplicate concentrations of 0.5 g/L; 1 g/L; and 2 g/L in growing media for 4 h, 24 h, 48 h, and 72 h. Following treatment, cells were incubated with 0.5 g/L MTT in growing media for 1 h. After media was removed, DMSO (Sigma-Aldrich) was added to the cells. Absorbance was measured at 570 nm (signal) and 690 nm (background).

2.7. Determination of the Antimicrobial Activity

The antimicrobial screening of two Cistus aqueous extracts was carried out using disc-diffusion and broth microdilution assays according to the guidelines of the Clinical Laboratory Standards Institute [50,51].
For preliminary screening, a disc diffusion assay was employed. Briefly, an aliquot of 100 µL of suspension of the mid-exponentially grown bacteria containing 106 CFU/mL of cells, and 0.5–2.5 × 103 CFU/mL of C. albicans spores were plated on MHA (Biolife) and SDA (Biolife). The 6 mm sterile filter discs (BectonDickinson, Franklin Lakes, NJ, USA) were individually loaded with 50 µL of the stock solution of Cistus aqueous extracts (0.1 g/mL) equivalent to a final concentration of 0.005 g/disc and then placed on the agar surface previously inoculated with the microbial strains. Tetracycline (30 µg) and amphotericin B (10 µg) discs were used as positive controls. The plates were incubated for 24 h at 37 °C. The antimicrobial activity was determined by measuring the diameters of the inhibition zones in millimeters. Assays were done in triplicate and the obtained values expressed as mean ± standard error (SE).
For the microdilution assays, experiments were performed in 96-well microtiter plates with serial dilutions of extracts ranging from 32 to 0.03125 mg/mL. The mid-exponentially grown bacterial cultures were adjusted spectrophotometrically to achieve optical densities corresponding to 1 × 106 CFU/mL, and 0.5–2.5 × 103 CFU/mL in case of C. albicans. After 24 h of incubation at 37 °C, the minimal inhibitory concentration (MIC) was recorded as the lowest concentration of the extract showing no visually detectable microbial growth in the wells.
For C. albicans, the corresponding aliquots from the wells were plated on SDA and incubated for 24 h. After visual inspection and colony counting, MIC90 endpoints were recorded as the lowest concentration, inhibiting 90% of fungal growth when compared to the control-strain grown without the extract.

2.8. Microbial Strains

The antimicrobial activity of aqueous Cistus extracts were assayed against eleven microbial strains, including the most emerging Gram-positive and Gram-negative pathogens: the type strains from the American Type Culture Collection (ATCC, Rockville, MD, USA) and the multidrug-resistant (MDR) hospital strains of Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa. The MDR strains demonstrated resistance to antimicrobial agents from at least three different classes, particularly to the β-lactam family of antibiotics as a result of β-lactamase(s) production (Table 1). The antibiotic susceptibility testing was assessed by Etest (AB Biodisk, Solna, Sweden) and the VITEK 2 system (bioMérieux, Marcy-l’Étoile, France). The molecular detection of genes mediating the β-lactam resistance phenotype was exerted as previously published [52]. Antibacterial screening also included the environmental strains of foodborne pathogens Bacillus cereus and Clostridium perfringens. The antifungal activity was assessed on the environmental strain of opportunistic pathogenic yeast Candida albicans (Table 2). All the microbial strains were stored at −80 °C in glycerol stocks and sub-cultured on tryptic soy agar (Biolife, Milan, Italy) or Sabouraud dextrose agar (Biolife, Milan, Italy) in case of fungi prior to testing.

3. Results and Discussion

3.1. UPLC-MS/MS Phytochemical Analysis of Plant Extracts

The preliminary phytochemical analysis of two Cistus aqueous extracts were performed using UPLC-MS/MS. Results of 43 chemical compounds along with their retention time, precursor ion, product ion, collision energy, and ionization mode optimized for each phenolic compound are presented in Table 1. A total of 43 polyphenolic compounds were identified, using standards where available. If a commercial standard was not available, quantification was performed using the calibration curve of standards from the same phenolic group.
Based on UPLC-MS/MS profiling of two Cistus extracts, this study reveals that aqueous extracts of CC and CS represent a rich source of polyphenols with flavonol derivatives being the most abundant compounds in both samples. Two flavonol glycosides, myricetin-3-hexoside and myricetin-rhamnoside, predominate in CC aqueous extracts. Myricetin-3-hexoside predominates in CS too, while myricetin-rhamnoside is present in significantly smaller amounts. Besides these two dominant phenolic compounds, two of the studied species show general similarity in the content for most of the analyzed phenolics, except that CC has a significantly higher amount of myrcetin-rutinoside, myrcetin, quercetin-rhamnoside, and procyanidin B2, while CS has a higher amount of quercetin-3-glucoside, quercetin-pentoside, kaempferol-3-glucoside, kaempferol-3-rutinoside, kaempferol-rhamnosyl-hexoside, and gallic acid, when compared to CS.
The phenolic profile of both extracts comprises phenolic acids, flavanols, flavonol glycosides and flavonones, which is in accordance with results obtained by previous Cistus studies. This is the first study of the phenolic composition in C. certicus and C. salvifolius aqueous extracts and the presence of phenolic compounds is in accordance with previous studies related to phenolic content in other Cistus species [11,42,53,54,55,56].

3.2. Antioxidant Activity of Cistus Extracts

The total phenolic content and antioxidant activity of CC and CS were assessed using in vitro assays and the results for both extracts are shown in Table 2.
The higher phenolic content of CC (209.27 mg of GAE/g) and CS (161.09 mg of GAE/g) aqueous extracts were at least doubled than in the previous study on the same species from Syria [57]. Higher phenolic content of CC (209.27 mg of GAE/g) in respect to CS (161.09 mg of GAE/g) aqueous extract determined through the Folin-Ciocalteu reagent correlates to its higher antioxidant capacity observed with the DPPH method (IC50 values were 24.0 μg/mL and 29.5 μg/mL, respectively). Correlation between phenolic content and antioxidant activity measured by DPPH method is in accordance with the fact that both assays are based on the electron-transfer mechanism [58]. The results obtained for DPPH measurements correlate with the literature data reported for three Tunisian′s Cistus [59]. Similar findings are also reported for CC and CS extracts from Syria with IC50 values of 11 μg/mL and 19 μg/mL, respectively [57]. The FRAP assay showed no positive correlation between antioxidant activity and total phenolic content in this study. The results obtained for the FRAP measurements for CC and CS were 0.78 Eq mM Fe2+ for both extracts.
The values obtained for antioxidant measurements of two Cistus extracts using DPPH and FRAP are consistent with others reported for the Cistus genus [42]. Too few studies have reported and stressed CC and CS phenolic and antioxidant potential. However, Cistus species have already been identified as a promising source of natural antioxidant compounds from plants, and as a potential means of finding new sources of natural antioxidants, functional foods, and nutraceuticals [60,61].
In this study, the water was used as an extraction solvent to isolate hydrophilic antioxidants from the plants. Certainly, for use as food and nutraceuticals, aqueous plant extracts are nutritionally more relevant and would have obvious advantages in relation to certification and safety [60]. Comparing to other studies of Cistus species, this study of CC and CS aqueous extracts from Croatia showed a significant amount of determined phenolics. Since they are involved in many biological processes, the phenolic and flavonoids compounds have been the subject of numerous studies, especially in strategies of reducing or preventing the generation of oxidative stress. Besides their direct antioxidant effects, a review of recent studies on the protective role of polyphenols on human health reveals the importance of their indirect effect, too. Along with antioxidant activity, phenolic compounds have been recognized by their antimicrobial and antiproliferative properties [62,63].

3.3. Antiproliferative Activity of Cistus Extracts

Antiproliferative activity of CC and CS aqueous extracts were tested on human lung cancer (A549) cell line after 4 h, 24 h, 48 h, and 72 h treatment, using the MTT assay. Both CC and CS aqueous extracts show a dose- and time-dependent antiproliferative activity against tested cell line (Figure 1).
Both Cistus extracts showed very similar antiproliferative activity on the lung cancer cell line A549. After 72 h of treatment with CC and CS aqueous extracts in concentration of 0.5 g/L, the cell viability was 37% and 50% respectively, compared to non-treated cells (100% of viability, cell survival = 1, Figure 1). The highest antiproliferative activity of 90% inhibition was demonstrated by CC extract after 72 h treatment for concentration of 2 g/L.
Natural herbal extracts have already been identified as a potential dietary source of polyphenols in the prevention/treatment of human chronic diseases such as cancer [64]. The studies of anticancer activity of several tea extracts have reported CC50 values within the range 0.1–0.5 mg/mL [65]. It has been reported that Cistaceae aqueous extracts containing ellagitannins showed a significant capacity to inhibit the proliferation on several cancer cell lines [66]. The results obtained in this study are in correlation with previous studies related to cytotoxicity of Cistus extracts, with reported CC50 values around 0.55 mg/mL [66,67]. The chemo-preventive and anti-tumor effects of a polyphenols-rich diet used to be associated with their ability to inhibit reactive oxygen species (ROS). However, the recent studies bring the evidence that their direct antiproliferation activity is related to modulation of uncontrolled proliferation pathways or proto-oncogene expression via multiple mechanisms [68]. Based on the fact that both extracts are rich in flavonol derivatives and that they exhibit similar antiproliferative activity, it can be assumed that these compounds can be attributed to this effect. This assumption can be supported by other studies that show the antiproliferative activity of flavonoids [69,70,71].
The present preliminary study, for the first time, reported antiproliferative activity of the Cistus aqueous extracts on the A549 lung cancer cell line. The concentration of 0.5 mg/mL of two Cistus extracts showing the antiproliferative effect against A549 cancer cells might be very significant to support further studies.

3.4. Antimicrobial Potential of Cistus Extracts

This study has assessed the antimicrobial potential of the CC and CS aqueous extracts, using disc-diffusion and broth microdilution assays, targeting the opportunistic pathogens that are commonly associated with food poisoning as well as urinary, respiratory tract, blood stream, and wound infections in humans. The study also included the antibiotic-susceptible ATCC strains as well as multiple resistant clinical strains of S. aureus (MRSA-1), K. pneumoniae, A. baumannii, and P. aeruginosa to observe the possible difference in the activity of extracts towards the strains of the same bacterial species but different antibiotic susceptibility/resistance patterns.
Overall, the antimicrobial assays revealed that fungi were in general more sensitive to both Cistus aqueous extracts in comparison to the bacteria as presented in Table 3 and Table 4.
As shown in Table 3, CS extract exhibited very good antifungal activity against opportunistic yeast C. albicans (MIC 125 µg/mL and inhibition zone of 23.2 ± 1.2 mm). On the other hand, MIC values for both extracts against bacterial strains were recorded in the range of 250 to 2000 µg/mL, respectively. The measured inhibition zones for 5 mg of each extract/disc ranged from 10.0 ± 0.5 to 29.0 ± 0.8 mm. It is important to note that, in general, the two extracts showed very similar activity towards the same bacterial species. However, slightly better activity of CC was evident against S. aureus ATCC 29213 and P. aeruginosa ATCC 27853 (MICs 500 µg/mL). Among the tested bacterial strains, the most potent activity was observed against A. baumannii FSST-20 clinical isolate (MIC 250 µg/mL) for both extracts. Notably, this particular multiple resistant strain as well as the P. aeruginosa FSST-21 and the MRSA-1 strain were found to be more sensitive and had a 2-fold reduction in MIC of at least one extract when compared to the antibiotic-susceptible ATCC strains. This finding indicates the potential of Cistus extracts to act against both Gram-negative and Gram-positive multiple resistant opportunistic pathogens that cause infections, which are therefore hard to treat by conventional antibiotics. The most promising activity was recorded by the disc-diffusion assay against the MRSA-1 strain, which was inhibited by 29.0 ± 0.8 and 25.0 ± 1.0 mm at the concentration of 5 mg/disc for CC and CS aqueous extracts, respectively.
The antimicrobial effect of various extracts, including the aqueous ones, derived from plants of the Cistaceae family have long been in the focus of scientific interest [72]. Most recently, C. incanus herbal tea has demonstrated antibacterial and anti-adherent effects against Streptococcus mutans, a causative agent of caries disease [34]. Moreover, C. ladanifer and C. populifolius leaf aqueous extracts were found to be active against S. aureus and E. coli (MIC50 from 0.123 to 0.9 mg/mL) [66]. However, there are only few reports on the investigation of the antimicrobial potency of CC and CS aqueous extracts. Previously, aqueous extracts obtained via spray-drying from Spanish CS were found to exhibit significant bacteriostatic and bactericidal effects against S. aureus CECT 59 strain (MIC50 52 ± 18 µg/mL) [14]). But, the comparison with the data obtained in our study could not be given due to the difference in extraction method as well as the S. aureus strain used. Moreover, Güvenç et al. carried out an antimicrobial study on various extracts from Turkish Cistus species by the disc-diffusion method, and found very weak activity of CC and CS aqueous extracts against S. aureus ATCC 29,213 and B. cereus 1122 strains [44]. In addition, no effect was observed against P. aeruginosa and C. albicans in this study.

4. Conclusions

To our knowledge, this work marks the first study of the phenolic components from aqueous extracts of two Croatian Cistus species: C. criticus and C. salviifolius. In summary, the results of this study of two Cistus species from Croatia reveal that both extracts possess prominent antioxidant, antimicrobial, and antiproliferative potential. Preliminary results of cytotoxic activity were obtained and future works have to be done to confirm the safety of these extracts. UPLC-MS/MS data obtained from this study indicate that two Cistus extracts are abundant sources of bioactive compounds—polyphenols. Additional studies are also needed to characterize and trace the active compounds with the biological activities of the extracts. The herbal and tea infusions may be an effective auxiliary to prevent or treat cancers, and further study on the precise mechanisms responsible for the antiproliferative activities of these herbal and tea infusions is still required. Altogether, the obtained results highlight the importance of Cistus as a promising source of biologically active phytochemical compounds.

Author Contributions

I.C. writing–original draft preparation, plant sample collection, laboratory work, analysis of the data. A.M. and N.I. contributed to biological studies, O.P. contributed to analysis of data and critical reading of the manuscript, Z.Z. assessed chemical analysis of the samples, V.Č.Č. contributed in study design, M.R. designed study, supervised the laboratory work, writing–original/critical reading of the manuscript, review and editing. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the Croatian Science Foundation [HRZZ-IP-2014-09-6897].

Acknowledgments

We thank our botanist Jure Kamenjarin for determination of botanical material.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Mabberley, D.J. The Plant, 2nd ed.; Cambridge University Press: Cambridge, UK, 1997; p. 680. [Google Scholar]
  2. Petropoulos, S.A.; Karkanis, A.; Martins, N.; Ferreira, I.C. Halophytic herbs of the Mediterranean basin: An alternative approach to health. Food Chem. Toxicol. 2018, 114, 155–169. [Google Scholar] [CrossRef]
  3. Papaefthimiou, D.; Papanikolaou, A.; Falara, V.; Givanoudi, S.; Kostas, S.; Kanellis, A. Genus Cistus: A model for exploring labdane-type diterpenes’ biosynthesis and a natural source of high value products with biological, aromatic, and pharmacological properties. Front. Chem. 2014, 2, 35. [Google Scholar] [CrossRef]
  4. Alkofahi, A.; Al-Khalil, S. Mutagenic and Toxic Activity of some Jordanian Medicinal Plants. Int. J. Pharmacogn. 1995, 33, 61–64. [Google Scholar] [CrossRef]
  5. Bouamama, H.; Villard, J.; Benharref, A.; Jana, M. Antibacterial and antifungal activities of Cistus incanus and C. monspeliensis leaf extracts. Therapie 2000, 54, 731–733. [Google Scholar]
  6. Attaguile, G.; Russo, A.; Campisi, A.; Savoca, F.; Acquaviva, R.; Ragusa, N.; Vanella, A. Antioxidant activity and protective effect on DNA cleavage of extracts from Cistus incanus L. and Cistus monspeliensis L. Cell Biol. Toxicol. 2000, 16, 83–90. [Google Scholar] [CrossRef]
  7. Rebensburg, S.; Helfer, M.; Schneider, M.; Koppensteiner, H.; Eberle, J.; Schindler, M.; Gürtler, L.G.; Brack-Werner, R. Potent in vitro antiviral activity of Cistus incanus extract against HIV and Filoviruses targets viral envelope proteins. Sci. Rep. 2016, 6, 20394. [Google Scholar] [CrossRef]
  8. Sayah, K.; Chemlal, L.; Marmouzi, I.; El Jemli, M.; Cherrah, Y.; Faouzi, M.E.A. In vivo anti-inflammatory and analgesic activities of Cistus salviifolius (L.) and Cistus monspeliensis (L.) aqueous extracts. S. Afr. J. Bot. 2017, 113, 160–163. [Google Scholar] [CrossRef]
  9. Wannes, W.A.; Tounsi, M.; Marzouk, B. A review of Tunisian medicinal plants with anticancer activity. J. Complement. Integr. Med. 2017, 15, 1–35. [Google Scholar] [CrossRef]
  10. Bereksi, M.S.; Hassaine, H.; Bekhechi, C.; Abdelouahid, D.E. Evaluation of Antibacterial Activity of some Medicinal Plants Extracts Commonly Used in Algerian Traditional Medicine against some Pathogenic Bacteria. Pharmacogn. J. 2018, 10, 507–512. [Google Scholar] [CrossRef]
  11. Jeszka-Skowron, M.; Zgoła-Grześkowiak, A.; Frankowski, R. Cistus incanus a promising herbal tea rich in bioactive compounds: LC–MS/MS determination of catechins, flavonols, phenolic acids and alkaloids—A comparison with Camellia sinensis, Rooibos and Hoan Ngoc herbal tea. J. Food Compos. Anal. 2018, 74, 71–81. [Google Scholar] [CrossRef]
  12. Attaguile, G.; Perticone, G.; Mania, G.; Savoca, F.; Pennisi, G.; Salomone, S. Cistus incanus and Cistus monspeliensis inhibit the contractile response in isolated rat smooth muscle. J. Ethnopharmacol. 2004, 92, 245–250. [Google Scholar] [CrossRef] [PubMed]
  13. Bouamama, H.; Noel, T.; Villard, J.; Benharref, A.; Jana, M. Antimicrobial activities of the leaf extracts of two Moroccan cistus L. species. J. Ethnopharmacol. 2006, 104, 104–107. [Google Scholar] [CrossRef] [PubMed]
  14. Tomás-Menor, L.; Morales-Soto, A.; Barrajón-Catalán, E.; Roldan-Segura, C.M.; Segura-Carretero, A.; Micol, V. Correlation between the antibacterial activity and the composition of extracts derived from various Spanish Cistus species. Food Chem. Toxicol. 2013, 55, 313–322. [Google Scholar] [CrossRef] [PubMed]
  15. Vitali, F.; Pennisi, G.; Attaguile, G.; Savoca, F.; Tita, B. Antiproliferative and cytotoxic activity of extracts from Cistus incanus L. and Cistus monspeliensis L. on human prostate cell lines. Nat. Prod. Res. 2011, 25, 188–202. [Google Scholar] [CrossRef]
  16. Ustun, O.; Berrin-Ozcelik, T.B. Bioactivities of ethanolic extract and its fractions of Cistus laurifolius L. (Cistaceae) and Salvia wiedemannii Boiss. (Lamiaceae) species. Pharmacogn. Mag. 2016, 12, S82–S85. [Google Scholar] [CrossRef]
  17. Attaguile, G. Gastroprotective effect of aqueous extract of Cistus incanus L. in rats. Pharmacol. Res. 1995, 31, 29–32. [Google Scholar] [CrossRef]
  18. Venditti, A.; Bianco, A.; Bruno, M.; Ben Jemia, M.; Nicoletti, M. Phytochemical study of Cistus libanotis L. Nat. Prod. Res. 2014, 29, 189–192. [Google Scholar] [CrossRef]
  19. Politeo, O.; Maravić, A.; Burčul, F.; Carev, I.; Kamenjarin, J. Phytochemical Composition and Antimicrobial Activity of Essential Oils of Wild Growing Cistus species in Croatia. Nat. Prod. Commun. 2018, 13, 771–774. [Google Scholar] [CrossRef]
  20. Fernández-Calvet, A.; Euba, B.; Caballero, L.; Díez-Martínez, R.; Menendez, M.; Ortiz-De-Solorzano, C.; Leiva, J.; Micol, V.; Barrajón-Catalán, E.; Garmendia, J. Preclinical Evaluation of the Antimicrobial-Immunomodulatory Dual Action of Xenohormetic Molecules against Haemophilus influenzae Respiratory Infection. Biomolecules 2019, 9, 891. [Google Scholar] [CrossRef]
  21. Umarusman, M.A.; Aysan, Y.; Özgüven, M. Investigation of the antibacterial effects of different plant extracts against pea bacterial leaf blight disease caused by pseudomonas syringae pv. pisi|Farkli Bitki Ekstraktlarinin Bezelye Bakteriyel Yaprak Yanikligina (Pseudomonas syringae pv. pisi) A. J. Tekirdag Agric. Fac. 2019, 16, 297–314. [Google Scholar]
  22. Vasiliki, G.; Charalampia, D.; Haralabos, K.C. In Vitro Antioxidant, Antithrombotic, Antiatherogenic and Antidiabetic Activities of Urtica dioica, Sideritis euboea and Cistus creticus Water Extracts and Investigation of Pasta Fortification with the Most Bioactive One. Curr. Pharm. Biotechnol. 2019, 20, 874–880. [Google Scholar] [CrossRef] [PubMed]
  23. Lahcen, S.A.; El Hattabi, L.; Benkaddour, R.; Chahboun, N.; Ghanmi, M.; Satrani, B.; Tabyaoui, M.; Zarrouk, A. Chemical composition, antioxidant, antimicrobial and antifungal activity of Moroccan Cistus creticus leaves. Chem. Data Collect. 2020, 26, 100346. [Google Scholar] [CrossRef]
  24. Ammendola, M.; Haponska, M.; Balik, K.; Modrakowska, P.; Matulewicz, K.; Kazmierski, L.; Lis, A.; Kozlowska, J.; Garcia-Valls, R.; Giamberini, M.; et al. Stability and anti-proliferative properties of biologically active compounds extracted from Cistus L. after sterilization treatments. Sci. Rep. 2020, 10, 1–10. [Google Scholar] [CrossRef]
  25. Jerónimo, E.; Soldado, D.; Sengo, S.; Francisco, A.; Fernandes, F.; Portugal, A.P.; Alves, S.P.; Santos-Silva, J.; Bessa, R.J. Increasing the α-tocopherol content and lipid oxidative stability of meat through dietary Cistus ladanifer L. in lamb fed increasing levels of polyunsaturated fatty acid rich vegetable oils. Meat Sci. 2020, 164, 108092. [Google Scholar] [CrossRef] [PubMed]
  26. Mahmoudi, H.; Aouadhi, C.; Kaddour, R.; Gruber, M.; Zargouni, H.; Zaouali, W.; Hamida, N.B.; Nasri, M.B.; Ouerghi, Z.; Hosni, K. Comparison of antioxidant and antimicrobial activities of two cultivated cistus species from Tunisia|Comparação das atividades antioxidante e antimicrobiana de duas espécies cultivada de cistus da Tunísia. Biosci. J. 2016, 32, 226–237. [Google Scholar] [CrossRef]
  27. Mikulec, A.; Kowalski, S.; Makarewicz, M.; Skoczylas, Ł.; Tabaszewska, M. Cistus extract as a valuable component for enriching wheat bread. LWT 2020, 118, 108713. [Google Scholar] [CrossRef]
  28. Lisiecka, K.; Wójtowicz, A.; Dziki, D.; Gawlik-Dziki, U. The influence of Cistus incanus L. leaves on wheat pasta quality. J. Food Sci. Technol. 2019, 56, 4311–4322. [Google Scholar] [CrossRef]
  29. Stępień, A.E.; Gorzelany, J.; Matłok, N.; Lech, K.; Figiel, A. The effect of drying methods on the energy consumption, bioactive potential and colour of dried leaves of Pink Rock Rose (Cistus creticus). J. Food Sci. Technol. 2019, 56, 2386–2394. [Google Scholar] [CrossRef]
  30. Viapiana, A.; Konopacka, A.; Waleron, K.; Wesolowski, M. Cistus incanus L. commercial products as a good source of polyphenols in human diet. Ind. Crop. Prod. 2017, 107, 297–304. [Google Scholar] [CrossRef]
  31. Chebli, Y.; El Otmani, S.; Chentouf, M.; Hornick, J.-L.; Bindelle, J.; Cabaraux, J.F.; Chebli, Y. Foraging Behavior of Goats Browsing in Southern Mediterranean Forest Rangeland. Animals 2020, 10, 196. [Google Scholar] [CrossRef]
  32. Guerreiro, O.; Alves, S.P.; Soldado, D.; Cachucho, L.; Almeida, J.M.; Francisco, A.E.; Santos-Silva, J.; Bessa, R.J.; Jerónimo, E. Inclusion of the aerial part and condensed tannin extract from Cistus ladanifer L. in lamb diets—Effects on growth performance, carcass and meat quality and fatty acid composition of intramuscular and subcutaneous fat. Meat Sci. 2020, 160, 107945. [Google Scholar] [CrossRef] [PubMed]
  33. Mignacca, S.A.; Mucciarelli, M.; Colombino, E.; Biasibetti, E.; Muscia, S.; Amato, B.; Presti, V.D.M.L.; Vazzana, I.; Galbo, A.; Capucchio, M.T. Cistus salviifolius Toxicity in Cattle. Veter. Pathol. 2019, 57, 115–121. [Google Scholar] [CrossRef] [PubMed]
  34. Wittpahl, G.; Kölling-Speer, I.; Basche, S.; Herrmann, E.; Hannig, M.; Speer, K.; Hannig, C. The Polyphenolic Composition of Cistus incanus Herbal Tea and Its Antibacterial and Anti-adherent Activity against Streptococcus mutans. Planta Med. 2015, 81, 1727–1735. [Google Scholar] [CrossRef] [PubMed]
  35. Dimas, K.; Demetzos, C.; Marsellos, M.; Sotiriadou, R.; Malamas, M.; Kokkinopoulos, D. Cytotoxic Activity of Labdane Type Diterpenes Against Human Leukemic Cell Lines in vitro. Planta Med. 1998, 64, 208–211. [Google Scholar] [CrossRef] [PubMed]
  36. Raimundo, J.R.; Frazão, D.F.; Domingues, J.L.; Quintela-Sabarís, C.; Dentinho, T.P.; Anjos, O.; Alves, M.N.; Delgado, F. Neglected Mediterranean plant species are valuable resources: The example of Cistus ladanifer. Planta 2018, 248, 1351–1364. [Google Scholar] [CrossRef] [PubMed]
  37. El Euch, S.K.; Bouajila, J.; Bouzouita, N. Chemical composition, biological and cytotoxic activities of Cistus salviifolius flower buds and leaves extracts. Ind. Crop. Prod. 2015, 76, 1100–1105. [Google Scholar] [CrossRef]
  38. Riehle, P.; Vollmer, M.; Rohn, S. Phenolic compounds in Cistus incanus herbal infusions—Antioxidant capacity and thermal stability during the brewing process. Food Res. Int. 2013, 53, 891–899. [Google Scholar] [CrossRef]
  39. Rebaya, A.; Belghith, S.I.; Cherif, J.K.; Trabelsi-Ayadi, M. Total phenolic compounds and antioxidant potential of rokrose (Cistus salviifolius) leaves and flowers grown in Tunisia. Int. J. Pharmacogn. Phytochem. Res. 2016, 8, 327–331. [Google Scholar]
  40. El Menyiy, N.; Al-Waili, N.; El-Haskoury, R.; Bakour, M.; Zizi, S.; Al-Waili, T.; Lyoussi, B. Potential effect of Silybum marianum L. and Cistus ladaniferus L. extracts on urine volume, creatinine clearance and renal function. Asian Pac. J. Trop. Med. 2018, 11, 393. [Google Scholar] [CrossRef]
  41. Gürbüz, P.; Demirezer, L.Ö.; Güvenalp, Z.; Kuruüzüm-Uz, A.; Kazaz, C. Isolation and Structure Elucidation of Uncommon Secondary Metabolites from Cistus salviifolius L. Rec. Nat. Prod. 2015, 9, 175–183. [Google Scholar]
  42. Gori, A.; Ferrini, F.; Marzano, M.C.; Tattini, M.; Centritto, M.; Baratto, M.C.; Pogni, R.; Brunetti, C. Characterisation and Antioxidant Activity of Crude Extract and Polyphenolic Rich Fractions from C. incanus Leaves. Int. J. Mol. Sci. 2016, 17, 1344. [Google Scholar] [CrossRef] [PubMed]
  43. Kalpoutzaki, E.; Aligiannis, N.; Mitaku, S.; Harvala, C.; Skaltsounis, L. New Semisynthetic Antimicrobial Labdane-Type Diterpenoids Derived from the Resin “Ladano” of Cistus creticus. Z. Naturforschung C 2001, 56, 49–52. [Google Scholar] [CrossRef] [PubMed]
  44. Güvenç, A.; Yıldız, S.; Özkan, A.M.; Erdurak, C.S.; Coskun, M.; Yılmaz, G.; Okuyama, T.; Okada, Y.; Yildiz, S.; Yilmaz, G. Antimicrobiological Studies on Turkish Cistus. Species. Pharm. Biol. 2005, 43, 178–183. [Google Scholar] [CrossRef]
  45. Garofulić, I.E.; Zorić, Z.; Pedisić, S.; Brnčić, M.; Dragović-Uzelac, V. UPLC-MS2 Profiling of Blackthorn Flower Polyphenols Isolated by Ultrasound-Assisted Extraction. J. Food Sci. 2018, 83, 2782–2789. [Google Scholar] [CrossRef]
  46. Abu-Reidah, I.M.; Ali-Shtayeh, M.S.; Jamous, R.M.; Arráez-Román, D.; Segura-Carretero, A. HPLC-DAD-ESI-MS/MS screening of bioactive components from Rhus coriaria L. (Sumac) fruits. Food Chem. 2015, 166, 179–191. [Google Scholar] [CrossRef]
  47. Brand-Williams, W.; Cuvelier, M.; Berset, C. Use of a free radical method to evaluate antioxidant activity. LWT 1995, 28, 25–30. [Google Scholar] [CrossRef]
  48. Benzie, I.; Strain, J. The Ferric Reducing Ability of Plasma (FRAP) as a Measure of “Antioxidant Power”: The FRAP Assay. Anal. Biochem. 1996, 239, 70–76. [Google Scholar] [CrossRef]
  49. Singleton, S.; Rossi, J.A.; Singleton, V.L.; Singleton, V.; Rossi, J.; Singleton, V.I. Colorimetry of total phenolic with phosphomolybdic—Photsphotunstic acid reagents. Am. J. Vitic. 1965, 16, 144–158. [Google Scholar]
  50. Clinical and Laboratory Standards Institute. Reference Method for Broth Dilution Antifungal Susceptibility Testing of Filamentous Fungi: Approved Standard; Clinical and Laboratory Standards Institute: Wayne, PA, USA, 2008. [Google Scholar]
  51. Clinical and Laboratory Standards Institute. Performance Standard for Antimicrobial Susceptibility Testing—Approved Standard; Clinical and Laboratory Standards Institute: Wayne, PA, USA, 2012. [Google Scholar]
  52. Maravić, A.; Skocibusic, M.; Šamanić, I.; Fredotović, Ž.; Cvjetan, S.; Jutronić, M.; Puizina, J. Aeromonas spp. simultaneously harbouring blaCTX-M-15, blaSHV-12, blaPER-1 and blaFOX-2, in wild-growing Mediterranean mussel (Mytilus galloprovincialis) from Adriatic Sea, Croatia. Int. J. Food Microbiol. 2013, 166, 301–308. [Google Scholar] [CrossRef]
  53. Gürbüz, P.; Koşar, M.; Güvenalp, Z.; Kuruuzum-Uz, A.; Demirezer, L. Ömür Simultaneous determination of selected flavonoids from different Cistus species by HPLC-PDA. J. Res. Pharm. 2018, 22, 78–83. [Google Scholar] [CrossRef]
  54. Barrajón-Catalán, E.; Fernández-Arroyo, S.; Roldán, C.; Guillén, E.; Saura, D.; Segura-Carretero, A.; Micol, V. A systematic study of the polyphenolic composition of aqueous extracts deriving from several Cistus genus species: Evolutionary relationship. Phytochem. Anal. 2011, 22, 303–312. [Google Scholar] [CrossRef]
  55. Matłok, N.; Lachowicz, S.; Gorzelany, J.; Balawejder, M. Influence of Drying Method on Some Bioactive Compounds and the Composition of Volatile Components in Dried Pink Rock Rose (Cistus creticus L.). Molecules 2020, 25, 2596. [Google Scholar] [CrossRef] [PubMed]
  56. Gaweł-Bęben, K.; Kukula-Koch, W.; Hoian, U.; Czop, M.; Strzępek-Gomółka, M.; Antosiewicz, B. Characterization of Cistus × incanus L. and Cistus ladanifer L. Extracts as Potential Multifunctional Antioxidant Ingredients for Skin Protecting Cosmetics. Antioxidants 2020, 9, 202. [Google Scholar] [CrossRef]
  57. Ghalia, S.; Adawia, K.; Waed, A. Evaluation of radical scavenging activity, total phenolics and total flavonoids contents of Cistus species in Syria. Int. J. Pharmacogn. Phytochem. Res. 2016, 8, 1071–1077. [Google Scholar]
  58. Prior, R.L.; Wu, X.; Schaich, K. Standardized Methods for the Determination of Antioxidant Capacity and Phenolics in Foods and Dietary Supplements. J. Agric. Food Chem. 2005, 53, 4290–4302. [Google Scholar] [CrossRef] [PubMed]
  59. Nicoletti, M.; Toniolo, C.; Venditti, A.; Bruno, M.; Ben-Jemia, M. Antioxidant activity and chemical composition of three Tunisian Cistus: Cistus monspeliensis Cistus villosus and Cistus libanotis? Nat. Prod. Res. 2014, 29, 223–230. [Google Scholar] [CrossRef] [PubMed]
  60. Dudonné, S.; Vitrac, X.; Coutière, P.; Woillez, M.; Mérillon, J.-M. Comparative Study of Antioxidant Properties and Total Phenolic Content of 30 Plant Extracts of Industrial Interest Using DPPH, ABTS, FRAP, SOD, and ORAC Assays. J. Agric. Food Chem. 2009, 57, 1768–1774. [Google Scholar] [CrossRef]
  61. Piluzza, G.; Bullitta, S. Correlations between phenolic content and antioxidant properties in twenty-four plant species of traditional ethnoveterinary use in the Mediterranean area. Pharm. Biol. 2011, 49, 240–247. [Google Scholar] [CrossRef]
  62. Brunetti, C.; Di Ferdinando, M.; Fini, A.; Pollastri, S.; Tattini, M. Flavonoids as Antioxidants and Developmental Regulators: Relative Significance in Plants and Humans. Int. J. Mol. Sci. 2013, 14, 3540–3555. [Google Scholar] [CrossRef] [PubMed]
  63. Agati, G.; Brunetti, C.; Di Ferdinando, M.; Ferrini, F.; Pollastri, S.; Tattini, M. Functional roles of flavonoids in photoprotection: New evidence, lessons from the past. Plant Physiol. Biochem. 2013, 72, 35–45. [Google Scholar] [CrossRef] [PubMed]
  64. Costa, C.; Tsatsakis, A.; Mamoulakis, C.; Teodoro, M.; Briguglio, G.; Caruso, E.; Sarandi, E.; Margină, D.; Dardiotis, E.; Kouretas, D.; et al. Current evidence on the effect of dietary polyphenols intake on chronic diseases. Food Chem. Toxicol. 2017, 110, 286–299. [Google Scholar] [CrossRef] [PubMed]
  65. Friedman, M.; Mackey, B.E.; Kim, H.-J.; Lee, I.-S.; Lee, K.-R.; Lee, S.-U.; Kozukue, E.; Kozukue, N. Structure−Activity Relationships of Tea Compounds against Human Cancer Cells. J. Agric. Food Chem. 2007, 55, 243–253. [Google Scholar] [CrossRef] [PubMed]
  66. Barrajón-Catalán, E.; Fernández-Arroyo, S.; Saura, D.; Guillén, E.; Gutierrez, A.F.; Segura-Carretero, A.; Micol, V. Cistaceae aqueous extracts containing ellagitannins show antioxidant and antimicrobial capacity, and cytotoxic activity against human cancer cells. Food Chem. Toxicol. 2010, 48, 2273–2282. [Google Scholar] [CrossRef]
  67. Skorić, M.; Todorović, S.I.; Gligorijević, N.; Janković, R.; Živković, S.; Ristić, M.; Radulović, S. Cytotoxic activity of ethanol extracts of in vitro grown Cistus creticus subsp. Creticus L. on human cancer cell lines. Ind. Crop. Prod. 2012, 38, 153–159. [Google Scholar] [CrossRef]
  68. Ismail, T.; Calcabrini, C.; Diaz, A.R.; Fimognari, C.; Turrini, E.; Catanzaro, E.; Akhtar, S.; Sestili, P. Ellagitannins in Cancer Chemoprevention and Therapy. Toxins 2016, 8, 151. [Google Scholar] [CrossRef] [PubMed]
  69. Lopez-Lazaro, M.; Calderón-Montaño, J.M.; Morón, E.B.; Austin, C.A. Green tea constituents (−)-epigallocatechin-3-gallate (EGCG) and gallic acid induce topoisomerase I- and topoisomerase II-DNA complexes in cells mediated by pyrogallol-induced hydrogen peroxide. Mutagenesis 2011, 26, 489–498. [Google Scholar] [CrossRef] [PubMed]
  70. Calderón-Montaño, J.M.; Morón, E.B.; Pérez-Guerrero, C.; Lopez-Lazaro, M. A review on the dietary flavonoid kaempferol. Mini Rev. Med. Chem. 2011, 11, 298–344. [Google Scholar] [CrossRef]
  71. Semwal, D.K.; Semwal, R.B.; Combrinck, S.; Viljoen, A.M. Myricetin: A Dietary Molecule with Diverse Biological Activities. Nutrients 2016, 8, 90. [Google Scholar] [CrossRef]
  72. Abad, M.J.; Bedoya, L.M.; Bermejo, P. Chapter 14—Essential Oils from the Asteraceae Family Active against Multidrug-Resistant Bacteria A2—Kon, Mahendra Kumar RaiKateryna Volodymyrivna. In Fighting Multidrug Resistance with Herbal Extracts, Essential Oils and Their Components; Academic Press: Cambridge, MA, USA, 2013; pp. 205–221. Available online: http://www.sciencedirect.com/science/article/pii/B9780123985392000148 (accessed on 9 July 2020).
Figure 1. Dose-time response effect of C. creticus (a) and C. salviifolius (b) aqueous extracts on human lung cancer cell lines A549, using the MTT assay.
Figure 1. Dose-time response effect of C. creticus (a) and C. salviifolius (b) aqueous extracts on human lung cancer cell lines A549, using the MTT assay.
Life 10 00112 g001
Table 1. Mass spectrometric data, identification and quantification of phenolic compounds from aqueous extracts of C. creticus L. (CC) and C. salviifolius L. (CS).
Table 1. Mass spectrometric data, identification and quantification of phenolic compounds from aqueous extracts of C. creticus L. (CC) and C. salviifolius L. (CS).
CompoundRt, minCollision
Energy (V)
Ionization
Mode
Precursor
Ion (m/z)
Fragment
Ions (m/z)
IdentificationC. C. **
(mg/L)
C. S. **
(mg/L)
11.13110367193Feruloylquinic acid0.31 ± 0.020.07 ± 0.02
21.13510353119Caffeoylqunic acid derivative 10.16 ± 0.040.18 ± 0.28
31.16910324173Caffeoylquinic acid derivative 20.15 ± 0.060.05 ± 0.04
41.68610343191Galloylquinic acid1.16 ± 0.055.47 ± 0.49
51.69315+442.9139Epicatechin gallate *0.52 ± 0.030.23 ± 0.10
61.7110331169Monogalloyl glucose [42]0.66 ± 0.492.84 ± 0.22
71.75810169125Gallic acid *2.83 ± 0.1124.66 ± 0.99
82.50810495169Digalloylquinic acid0.37 ± 0.040.33 ± 0.11
92.97315+653303Quercetinacetylrutinoside0.41 ± 0.500.08 ± 0.01
103.99310 337163Coumaroilquinic acid derivative 12.48 ± 1.350.34 ± 0.47
114.01410+579427Procyanidin B2 *11.65 ± 0.313.83 ± 0.29
124.30510+291139Catechin *5.49 ± 0.260.57 ± 0.06
134.31110+291139Epicatechin *5.46 ± 0.050.52 ± 0.13
144.45910353119Chlorogenic acid *0.06 ± 0.010.02 ± 0.01
154.97415+597303Quercetin-pentosyl-hexoside0.04 ± 0.010.97 ± 0.15
165.01610179135Caffeic acid *0.36 ± 0.550.05 ± 0.02
175.54110337163Coumaroilquinic acid derivative 20.41 ± 0.010.39 ± 0.53
186.02515+459139Epigallocatechin gallate *0.23 ± 0.064.77 ± 0.70
196.0375+403271Apigenin-pentoside0.07 ± 0.040.03 ± 0.01
206.3625+627315Myricetin-rutinoside [53]31.23 ± 0.103.09 ± 0.19
216.47415+481319Myricetin-hexoside [42]937.44 ± 0.49564.13 ± 3.78
226.57110163119p-coumaric acid *0.25 ± 0.030.23 ± 0.05
237.1915+611303Ruthin *3.46 ± 0.060.99 ± 0.08
247.2375+465319Myricetin-rhamnoside [53]874.51 ± 0.463.13 ± 0.71
257.25425+319273Myricetin *87.14 ± 0.4228.27 ± 1.11
267.31510193134Ferulic acid *0.55 ± 0.390.07 ± 0.02
277.4525+465303.1Quercetin-3-glucoside *26.08 ± 0.2076.49 ± 1.50
287.56115+581287Kaempferol-pentosylhexoside0.03 ± 0.010.51 ± 0.11
297.6510+647169Trigalloylquinic acid0.24 ± 0.040.04 ± 0.02
307.9715+495319Myricetin-glucuronide0.76 ± 0.070.22 ± 0.08
318.0445+435303Quercetin-pentoside [54]6.02 ± 0.0254.88 ± 1.82
328.1195+449287Kaempferol-3-glucoside [54]15.05 ± 0.4120.56 ± 1.25
338.16415+625317Isorhamnetin-rutinoside0.33 ± 0.020.03 ± 0.02
348.20335+287153Luteolin *4.28 ± 0.235.46 ± 1.40
358.4225+449303Quercetin-rhamnoside [53]23.02 ± 1.720.56 ± 0.29
368.6515+419287Kaempferol-pentoside1.14 ± 0.125.81 ± 0.79
378.72315+637287Kaempferol-acetylrutinoside0.03 ± 0.010.05 ± 0.03
388.7810+507303Quercetin-acetylhexoside0.13 ± 0.050.36 ± 0.56
399.5415+491287Kaempferol-acetylhexoside0.05 ± 0.030.24 ± 0.15
4010.6105+433287Kaempferol-rhamnoside1.28 ± 0.070.44 ± 0.30
4110.84315+595287Kaempferol-3-rutinoside *4.56 ± 0.3812.58 ± 0.80
4210.84615+595287Kaempferol-rhamnosyl-hexoside7.07 ± 2.8913.12 ± 1.64
4311.47530+271153Apigenin *0.35 ± 0.390.05 ± 0.03
* Identification confirmed using authentic standards. ** Concentration of individual phenolic compounds are expressed as mg per L of sample, as mean value ± standard deviation.
Table 2. Total phenolic content and antioxidant activities of the Cistus aqueous extracts.
Table 2. Total phenolic content and antioxidant activities of the Cistus aqueous extracts.
Antioxidant Assay (units)C. creticusC. salvifoliiusAscorbic Acid
Folin-Ciocalteu (mg GAE a)209.27 ± 18.5161.09 ± 7.2nd
DPPH b (mg/mL)0.52 ± 0.030.62 ± 0.040.02 ± 0.01
FRAP (equivalents of Fe2+ mM c)0.78 ± 0.020.78 ± 0.065.57 ± 0.13
a Gallic acid equivalents (mg of GAE/g); b IC50 (mg/mL); c Sample concentration 1 g/L; Values are represented as mean’s ± SD (n = 3).
Table 3. Antibiotic resistance profile and genotypic features of the multiple resistant and β-lactamases-producing clinical strains used in this study.
Table 3. Antibiotic resistance profile and genotypic features of the multiple resistant and β-lactamases-producing clinical strains used in this study.
Strain No.Speciesβ-lactam Resistance
Phenotype
MIC (µg/mL) of Selected Antimicrobial Agents aβ-Lactamase(s) ProducedNon β-Lactam Resistance
Phenotype
AMPCAZIPMGENTETOXESBLsMBLsOthers
FSST 02Klebsiella pneumoniaeAMP, SAM, PIP, TZP, TIC, CAZ, CTX, ATM>1024 (R)512 (R)0.5 (S)8 (R)128 (R)NASHV-12, CTX-M-15-ACC-1, TEM-1CIP, GEN, TET, SXT
FSST 20Acinetobacter baumanniiAMP, SAM, PIP, TZP, TIC, CTX, CAZ, FEP, ATM, IMP, MEM>1024 (R)>256 (R)16 (R)16 (R)64 (R)NA-SIM-1-GEN, TOB, AMK
FSST 21Pseudomonas aeruginosaAMP, SAM, PIP, TZP, TIC, CTX, CAZ, FEP, ATM, IMP, MEM>1024 (R)>16 (R)>8 (R)>8 (R)>8 (R)NA-GIM-1-CIP, GAT, LVX, AMK, GEN, TOB, NET, TET, SXT
MRSA-1Staphylococcus aureusOX, PNANANA>16 (R)<1 (S)>4 (R)Modification of PBP (mecA)CC, CIP, E, GEN, MFX
a MIC values were evaluated according to the breakpoints established by CLSI (2012). For S. aureus MRSA-1 breakpoint interpretation was done according to EUCAST (2012) guidelines. Abbreviations: R, resistant; S, susceptible; NA, not applicable; AMK, amikacin; AMP, ampicillin; ATM, aztreonam; CAZ, ceftazidime; CC, clindamycin; CIP, ciprofloxacin; CTX, cefotaxime, E, erythromycin; FEP, cefepime; GAT, gatifloxacin; GEN, gentamicin; IPM, imipenem; LVX, levofloxacin; MEM, meropenem; MFX, moxifloxacin; NET, netilmicin; OX, oxacillin; P, penicillin G; PIP, piperacillin; SAM, ampicillin-sulbactam; SXT, trimethoprim-sulfamethoxazole; TET, tetracycline; TIC, ticarcillin; TOB, tobramycin; TZP, piperacillin/tazobactam.
Table 4. Antimicrobial activity of the Cistus extracts against opportunistic pathogenic bacteria and fungi used in this study.
Table 4. Antimicrobial activity of the Cistus extracts against opportunistic pathogenic bacteria and fungi used in this study.
SpeciesStrain No.Inhibition Zone Diameter (mm) aMIC (µg/mL) c
C. creticus ExtractC. salviifolius
Extract
TET bC. creticus ExtractC. salviifolius
Extract
TET
Gram-positive bacteria
Staphylococcus aureusATCC 2921318.4 ± 1.115.2 ± 0.931.0 ± 0.550010000.5 (S)
Staphylococcus aureusMRSA-129.0 ± 0.825.0 ± 1.0NT500500<1 (S)
Bacillus cereusFSST-22 13.0 ± 0.513.0 ± 0.724.6 ± 0.6100010001 (S)
ClostridiumperfringensFSST-24 12.1 ± 0.512.4 ± 1.0NA1000500NA
Gram-negative bacteria
Klebsiella pneumoniaeATCC 1388317.4 ± 0.513.5 ± 1.021.3 ± 0.2100010001 (S)
Klebsiella pneumoniaeFSST-0210.5 ± 0.010.0 ± 0.56.0 ± 0.020002000128 (R)
Pseudomonas aeruginosaATCC 2785312.6 ± 0.910.3 ± 0.811.5 ± 0.41000100032 (R)
Pseudomonas aeruginosaFSST-2113.2 ± 0.411.0 ± 0.57.3 ± 0.35001000>16 (R)
Acinetobacter baumanniiATCC 1960617.1 ± 0.520.5 ± 1.517.3 ± 0.65005002 (S)
Acinetobacter baumanniiFSST-2023.0 ± 0.824.2 ± 0.06.0 ± 0.025025064 (R)
AMBMIC90AMB
Yeast
Candida albicansFSST-29 19.3 ± 0.823.2 ± 1.221.6 ± 1.75001251 (S)
a Diameters of the inhibition zones (in mm) were given for the 6 mm discs impregnated with final concentration of 5 mg/disc of the Cistus extracts. All assays were done in triplicate and values expressed as mean ± SD. b Discs of tetracycline (30 μg) and amphotericin B (10 μg) were used as standards for the disc diffusion assay. c For fungi, MIC90 values represent the lowest concentrations that inhibited 90% of fungal growth when compared to the growth without the addition of extracts. Abbreviations: AMB, amphotericin B; TET, tetracycline; NA, not applicable; NT, not tested; S, susceptible; R, resistant.
Back to TopTop