Background. The antimicrobial activity and effects of a phytocomplex consisting of Tropaeolum flos (T) and Salviae folium (S) extracts on the cytokine levels and transcription factors on dermal fibroblast BJ exposed to bacterial lipopolysaccharides were examined. Methods. In order to select the most optimal combination ratio of the two extracts for using in vitro, the physicochemical characterization of vegetal extract mixtures was performed and the antioxidant and antibacterial activities were evaluated on five different formulations of T : S, namely, 1 : 1, 1 : 2, 2 : 1, 3 : 1, and 1 : 3. The best combination of bioactive compounds with regard to antioxidant and antibacterial activities (T : S 1 : 2) was selected for in vitro evaluation of the anti-inflammatory effect. Human dermal fibroblast BJ cells were treated with two doses of the extract mixture and then exposed to bacterial lipopolysaccharides (LPS). The levels of the cytokines involved in inflammatory response, namely, interleukin- (IL-) 6, tumor necrosis factor- (TNF-) α, IL-31, and IL-33, were quantified by ELISA, and the expression of transcription factors, namely, signal transducer and activator of transcription (STAT) 3, nuclear factor kappa B (NFκB), and phosphorylated NFκB (pNFκB), were evaluated by western blot analysis. Results. The results have shown that the mixture of T : S 1 : 2 exhibited significant antibacterial effects on Staphylococcus aureus ATCC 25923. LPS exposure increased the cytokine levels in BJ cells and enhanced the NFκB expression. The pretreatment of BF cells exposed to LPS with the two doses of the extract mixture markedly inhibited the increase of IL-33 and TNF-α levels and amplified the NFκB expression and its activation, especially with the high dose. The low doses of the extract reduced NFκB expression but increased its activation. Conclusions. These experimental findings suggest that the mixture of T : S 1 : 2 can exert some protection against bacterial infections and inflammation induced by LPS in BJ cells being a good therapeutic option in related conditions associated with inflammation.
In the recent years, more and more studies focused on the pathogenetic mechanisms of atopic dermatitis (AD) and its treatment. Atopic dermatitis is one of the most common chronic inflammatory skin diseases whose incidence has increased considerably over the past decades, especially in industrialized countries. Thus, the prevalence of atopic dermatitis varies between 7% and 30% in children and between 1% and 10% in adults and evolves with a significant decrease in the quality of their life [1, 2].
AD is a complex condition with multifactorial aetiology, characterized by periods of exacerbation and remission with dry skin (xerosis), pruritus, and increased loss of transepidermal water . With all the remarkable progress made, the cause of AD has not been completely elucidated. Most studies consider that the disease appears as a result of the combined action of genetic features, barrier dysfunction, and environmental, immunological, and biochemical factors. The key factor in the pathogenesis of this disease is the alteration of the skin barrier due to loss of the functions of filaggrin, a structural protein, important for cornification and skin hydration . In addition, the affected skin is deficient in ceramide and has increased levels of endogenous proteolytic enzymes, responsible for transepidermic loss of water and alteration of cutaneous barrier function .
Several studies have shown the involvement of the imbalance of Th2 to Th1 cytokines and consequently the increased systemic response of Th2-type lymphocytes which initiate and maintain skin inflammation  and also enhance the hyperreactivity to environmental factors. Dysfunction of Th2 lymphocytes and cytokines released by them, namely, IL-4, IL-5, and IL-13, lead to increased immunoglobulin (Ig) E production, which amplifies the local inflammation and deteriorates the skin barrier function. In addition, Th17 and Th22 lymphocytes released the IL-17, IL-19, and IL-22 cytokines known to be involved in the pathogenesis of atopic dermatitis [7, 8]. As a response to changes in the skin barrier function, the keratinocytes secreted cytokines, such as stromal lymphopoietin, IL-25, and IL-33, which in turn activate Th2 lymphocytes and congenital lymphoid cells (ILC2) .
Several papers have suggested that new tissue-derived cytokines such as IL-33 have a significant role in AD [10, 11]. IL-33 belongs to the IL-1 family, and it is secreted by damaged tissues and activates Th2 lymphocytes, mast cells, basophils, and eosinophils to produce Th2-type cytokines [12, 13]. It seems that IL-33 would have a dual function: one by extracellular action, as a member of the IL-1 cytokine family, and the other by intracellular action, as nuclear factor that regulates gene expression [14–17].
Several evidences have shown an association of IL-31, a cytokine secreted by Th2 cells, mast cells, macrophages, and dendritic cells, with severe pruritus in inflammatory diseases including AD. The blocking of its expression mitigates the scratching behaviour in AD and chronic spontaneous urticaria [18, 19]. The expression of IL-31RA in human keratinocytes and macrophages is low in an unstimulated condition , and it is upregulated by interferon γ and toll-like receptor 2/toll-like receptor 1 agonists . Binding of IL-31 to its receptor determines the phosphorylation and activation of the mitogen-activated protein kinase (MAPK), c-Jun N-terminal kinase (JNK), and STAT pathways .
In recent years, there has been a growing demand for medicinal and aromatic herbs in the world due to the increased content in biologically active substances frequently used in the pharmaceutical, cosmetic, and food industries. Considerable research pointed out the potential effectiveness of natural compounds in biology and medicine due to their potent antioxidant, anti-inflammatory, and immunomodulatory properties [23–25]. These can be benefited by diet or through skin application, diminishing symptoms and inhibiting the development of various skin diseases without having the side effects of corticosteroids. Salvia officinalis L. (common sage) and Tropaeolum majus L. (garden nasturtium) are two of the most promising species for medical applications. Salvia officinalis L., the common sage or garden sage, is a perennial herb of the Lamiaceae family. Several studies demonstrated that Salvia officinalis L. had different biological activities which have antibacterial, virustatic, fungistatic, astringent, and antihydrotic effects [26–30]. The effects were due to the triterpenes oleanolic and ursolic acids and diterpene carnosol from a composition with anti-inflammatory properties and antiprotease and antimetastatic activities [31, 32]. In other Salvia species such as the S. verticillata subsp. amasica extract, the main compound identified was rosmarinic acid which explained the strong antioxidant activity .
Tropaeolum majus L., a garden nasturtium of the Tropaeolaceae family, is an annual plant originally from the Andes, Bolivia, Peru, and Colombia, known for the properties of the aerial part of Tropaeoli herba or the flowers of Tropaeoli flos [3, 34]. The Tropaeolum majus L. extract demonstrated antioxidant and anti-inflammatory activities due to its content of polyphenols, flavonoids, and ascorbic acid in different experimental models [35, 36].
Based on these data, the study is aimed at evaluating the physicochemical properties of the mixture of the two vegetal extracts obtained from crops harvested in Bihor County, Romania’s northern area, as well as evaluating the in vitro antioxidant capacity in order to assess their biological activities on human dermal fibroblast BJ exposed to bacterial lipopolysaccharides (LPS). It is known that LPS is a major component of the outer membrane of gram negative bacteria and a potent inductor of inflammation. Moreover, its bioactive moiety endotoxin can be measured in dust collected from homes. In addition, there are some papers which demonstrated a positive relationship between exposure to endotoxin and the high incidence of atopic dermatitis . Therefore, we used BJ cells exposed to LPS as a model to simulate in vitro atopic dermatitis.
We also tested the antimicrobial activity against different bacteria using the reference microbial strains and also the human clinical isolates from patients with infections or from the hospital environment. The evaluation of antimicrobial activity of the mixture of two extracts was important due to the increased incidence of infections in atopic dermatitis and the role played by infection as a trigger for disease exacerbation.
2. Materials and Methods
Galic acid, quercetin, 2,2-diphenyl-1-picryl-hydrazyl (DPPH free radical), 2,2-azino-bis (3-ethylbenzothiazoline-6-sulfonate) (ABTS), the ferric-reducing antioxidant power (FRAP), Folin Ciocâlteu reagents, and lipopolysaccharides from Escherichia coli O111:B4 were purchased from Sigma-Aldrich Chemicals GmbH (Germany). The Bradford reagent was from Merck KGaA (Darmstadt, Germany). ELISA tests for the evaluation of IL-6, IL-31, IL-33, TNF-α, and STAT3 were from R&D Systems Inc. (Minneapolis, MN, USA). The goat polyclonal IgG antibody for NFκB and pNFκB and the secondary antibody mouse anti-goat and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were bought from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). All the chemicals used were of analytical grade.
2.2. Plant Materials and Preparation of the Extract
Salvia officinalis L. and Tropaeolum majus L. plants were harvested from crops in Bihor County, Romania, in June–July 2018. The voucher specimens from both species were deposited in the Herbarium Pharmacy Department, University of Oradea (registered in the NYBG William and Lynda Steere Herbarium, code: UOP 05212 for Salvia officinalis L. and code: UOP 05073 for Tropaeolum majus L.). The plant material was dried after harvesting using an FDK 24 DW Gorenje dryer at 40°C for total moisture removal. The next step further, which was the vegetable product shredding, is one of the important factors in achieving the extractive preparations. By increasing the contact surface of the plant and solvent product, the extraction time is reduced and the extraction efficiency is increased. The degree of grinding of the plant products was chosen according to the European Pharmacopoeia (Ph Eur. 9th) and the Romanian Pharmacopoeia (FR X) and was passed through sieve I after a preliminary grinding with an electrical mill [38, 39].
The extracts were obtained by maceration at room temperature according to the European Pharmacopoeia (Ph. Eur. 9th) and Romanian Pharmacopoeia (FR X). The method was applied to both extracts of the flowers of Tropaeolum majus L. (denoted T) and extracts of the leaves of Salvia officinalis L. (denoted S). Each plant product was subjected to 30c ethyl alcohol maceration at room temperature (20°C) for 24 hours; the plant/30c alcohol ratio was 20% (/). After completion of the extraction, they were decanted and filtered using nylon filter paper, 100 μm.
2.3. Physicochemical Properties of the Mixture of Extracts
In previous papers, we performed the evaluation of the polyphenol and flavonoid content of each extract by reversed-phase HPLC (RP-HPLC) [35, 40]. The RP-HPLC analysis of the Tropaeolum majus L. extract has shown the following composition: in phenolic acids—gallic, caffeic, syringic, synapic, vanillic, p-coumaric, and ferulic; and in flavonoids—catechin hydrate, rutin trihydrate, naringenin, luteolin, quercetin dihydrate, epicatechin, and myricetin . The Salvia officinalis L. extract has an increased content of gallic acid, epicatechin, rutin, p-coumaric acid, luteolin, and quercetin.
Polyphenolic compounds and flavonoids have beneficial properties, acting as antioxidants in a biological system under oxidative stress conditions. In order to select the optimal combination ratio of the two extracts, several T : S combinations were used, namely, 1 : 1, 1 : 2, 2 : 1, 3 : 1, and 1 : 3. For this study, we evaluated the content of bioactive compounds based on their antioxidant properties demonstrated by in vitro methods.
2.3.1. Determination of the Content in Polyphenolic Compounds
Determination of the content in polyphenolic compounds was performed by the Folin-Ciocâlteu method, the results being expressed in gallic acid (GAE) equivalents (mg/mL). In order to achieve this determination, 100 μL of fluid extract was taken and mixed with 1750 μL distilled water, 200 μL of Folin-Ciocâlteu reagent (diluted 1 : 10 /), and 1000 μL of 15% Na2CO3 solution and then kept at room temperature, away from light, for two hours. Then, the absorbance was measured at a wavelength of 765 nm using a UV-Vis spectrophotometer. The calibration curve was linear for the concentration range of 0.1-0.5 mg/mL for gallic acid. The content of the total polyphenols in the extracts is expressed as mg equivalent of gallic acid (GAE)/g dry weight extract (DW) .
2.3.2. Determination of Total Flavonoids
Determination of total flavonoids was performed by the colourimetric method . On this line, 1 mL of sample was taken and mixed with 4 mL of distilled water and placed in a 10 mL volumetric flask. Then, 3 mL of 5% NaNO2 solution was added, and after 5 minutes, 0.3 mL of 10% AlCl3 solution was added. After a further 6 minutes, 2 mL of 1 M NaOH was added. The flask was quenched to the mark with distilled water, and the absorbance was read at 510 nm. The calibration curve was used using the quercetin standard. The equation of the calibration curve is (), where represents the absorbance and represents mg quercetin.
2.4. Evaluation of the Antioxidant Activity of the Mixture of Extracts
A number of analytical methods have been developed to determine the antioxidant activity of natural products that are generally based on the reaction between an antioxidant species and a chromogenic compound. The antioxidant capacity of the extracts was evaluated by the following methods: DPPH (2,2-diphenyl-1-picryl-hydrazyl), ABTS (2,2-azinobis (3-ethylbenzothiazoline-6-sulfonic acid), and ferric-reducing antioxidant power (FRAP).
2.4.1. DPPH Method
The DPPH method is a spectrophotometric method widely used to test the ability of compounds to remove free radicals or their ability to donate hydrogen. The activity of capturing 2,2-diphenyl-1-picryl-hydrazyl radicals (DPPH) was determined using the method proposed by Pallag et al. . Thus, at a volume of 100 mL of plant extract, 2900 mL of DPPH methanolic solution (80 mM) was added. The absorbance of the resulting solution was read at 515 nm after 5 minutes. The following equation was used to determine the DPPH inhibitory capacity (%): where is absorption blank and is sample absorbance at 515 nm.
2.4.2. ABTS Method
The ABTS method has been widely used to evaluate antioxidant activity and uses the method of Arnao et al. . Briefly, ABTS⁺ was produced by reacting the ABTS solution (7 mM) with 2.45 mM potassium persulphate, keeping the mixture in the dark at room temperature for 16 hours. The stock solution of ABTS was diluted to obtain an absorbance of at 734 nm. After the addition of 25 μL extract in 2.5 mL of diluted ABTS⁺, the mixture was homogenized very well (using Vortex) for 30 seconds and the interaction between antioxidants and ABTS•+ was determined spectrophotometrically at 734 nm, exactly 1 minute after homogenization. Trolox was used and a standard linear curve was obtained between 0.125 and 2 mmol/L Trolox. The ABTS value was obtained using the calibration curve equation: (), where is absorbance and is mmol Trolox equivalents.
2.4.3. Ferric-Reducing Antioxidant Power (FRAP) Method
The ferric-reducing antioxidant power (FRAP) method is a simple spectrophotometric method that tests the antioxidant potency of the samples taken in the study and is based on the reduction of the ferric tripyridyl-triazine complex to the ferrous tripyridyl triazine complex (Fe (III)-TPTZ) by a pH-reducing agent . Stock solutions include 300 mM acetate buffer; 270 mg of FeCl3·6H2O dissolved in 50 mL of distilled water; 150 mg of TPTZ; and 150 mL of HCl, dissolved in 50 mL of distilled water. The FRAP solution obtained was freshly prepared by mixing 50 mL of acetate buffer solution, 5 mL of FeCl3·6H2O solution, and 5 mL of TPTZ solution. The vegetable extracts (100 mL) were allowed to react with 500 mL of FRAP solution and 2 mL of distilled water for 1 hour, away from light. The final coloured product (ferric tripyridyl-triazine complex) was quantitated by absorption into the VIS at 595 nm. Trolox was used as the antioxidant positive control, and a standard linear curve of between 50 and 500 mmol/L of Trolox was obtained. The FRAP value was obtained using the equation based on the following calibration curve: (), where is absorbance and is mmol of Trolox equivalents.
2.5. Antimicrobial Activity
In vitro testing of the antimicrobial activity of the mixture of two plant extracts on bacteria was done by the diffusion method of Kirby-Bauer . We used two reference strains from the American Type Culture Collection (ATCC), namely, Staphylococcus aureus ATCC 25923 and Streptococcus pneumoniae ATCC 49619 and two wild strains, namely, Streptococcus pyogenes and Streptococcus agalactiae—isolated from human clinical cases. Culture media (Mueller-Hinton (Oxoid) for Staphylococci and Mueller-Hinton Agar sheep blood (BioMérieux) for Streptococci) were inoculated with standardized bacterial inoculum (0.5 McFarland units). After 10-15 minutes, 6 sterile filter papers (HiMedia Laboratories, Ref. SD067-5CT) 6 mm in diameter were placed on each Petri plate and 20 microliters of plant extract were impregnated into each disk. For each extract, the samples were worked in triplicate to minimize errors. We utilized standard penicillin disks (10 U, Oxoid) as a positive control, and paper disks smeared with distilled water (20 μL) as a negative control. After incubating the media at 37°C for 18 hours, the diameters of the inhibition zones were measured with a ruler and the arithmetic mean for each extract was calculated.
2.6. Cell Cultures
The assays were performed on normal human dermal fibroblast BJ (ATCC, Gaithersburg, Maryland USA). Cell culture medium was DMEM (Dulbecco’s modified Eagle’s medium), supplemented with 5% FBS (foetal bovine serum), antibiotics, and antimycotics; all reagents were purchased from Sigma-Aldrich Chemicals GmbH (Heidelberg, Germany).
2.6.1. Viability Assay
Cell survival was assessed through the colourimetric measurement of formazan, a coloured compound synthesized by viable cells, using the CellTiter 96® AQueous Nonradioactive Cell Proliferation Assay (Promega Corporation, Madison, USA). The dermal fibroblast BJ cultures were cultivated at a density of 10⁴/wells in 96-well plaques (TPP, Trasadingen, Switzerland) for 24 h, then exposed to the plant extracts in five different formulations, according to the following ratios between Tropaeolum majus (T) and Salvia officinalis extracts (S), respectively: (1) 1 : 1, (2) 1 : 2, (3) 2 : 1, (4) 3 : 1, and (5) 1 : 3. Concentrations ranging between 0 and -500 μg GAE/mL of polyphenols were prepared from each extract in medium immediately before use. The LPS impact on viability was also tested, similarly to the extracts by exposing BJ cells to different LPS concentrations, between 0 and 10 μg/mL. Cells were either exposed only to the extracts for 24 h, or, following the extracts’ exposure, cells were washed and further exposed to LPS (lipopolysaccharides from Escherichia coli) in a concentration of 10 μg/mL for an additional 24 h, then viability was measured colourimetrically, using an ELISA plate reader (Tecan, Männedorf, Switzerland) at 540 nm. The dose of 10 μg/mL for LPS was chosen based on the doses used in the literature including ex vivo models . All the experiments were done in triplicate. Untreated cell cultures were used as controls. Results are presented as OD540.
2.6.2. Cell Lysates
The BJ cells, seeded on Petri dishes at a density of 10⁴/cm², were exposed for 24 h to T : S 1 : 2 extract in concentration of 0.1 μg polyphenols/mL () and 0.01 μg polyphenols/mL () respectively, and then to LPS 10 μg/mL for an additional 24 h, or only extract or LPS. Untreated cells were used as controls. Cells were washed following exposure, and afterwards, lysates were prepared as previously described . Protein concentrations were determined by the Bradford method according to the manufacturer’s specifications (Bio-Rad, Hercules, CA, USA), using albumin bovine serum as standard. For all assays, the lysates were corrected by total protein concentration.
2.6.3. Inflammation Marker Assessment
Inflammation was assessed by the measurement of IL-31, IL-33, IL-6, TNF-α, and STAT3 levels using ELISA immunoassay kits from R&D Systems Inc. (Minneapolis, MN, USA). In addition, the expressions of transcription factor NFκB and its phosphorylated form (pNFκB) were evaluated by western blot analysis. For western blot, a 20 μg protein/lane was separated by electrophoresis on SDS-PAGE gels and then transferred to polyvinylidene difluoride membranes (Bio-Rad Mini-PROTEAN System from Bio-Rad) as previously described . Blots were blocked and then incubated with antibodies against NFκB, pNFκB p65 (Ser536) (93H1), and GAPDH and then further washed and incubated with corresponding secondary peroxidase-linked antibodies (Santa Cruz Biotechnology Inc.). The proteins were detected using the SuperSignal West Femto chemiluminescent substrate (Thermo Fisher Scientific, Rockford, IL, USA) and were then quantified using Quantity One Analysis Software (Bio-Rad).
2.7. Statistical Analysis
The statistical significance of the results was conducted by using one-way ANOVA, followed by Tukey’s multiple tests. All reported data were expressed as the mean of (SD), and a value lower than 0.05 was considered statistically significant.
3.1. Physicochemical Properties of the Mixture of Extracts
The polyphenols from the mixture of extracts were identified by comparing the data from the chromatogram of the extract with a chromatogram of a standard solution. Compound detection was performed at several wavelengths: 235, 255, 259, 260, 270, 274, 280, 285, 310, 320, and 345 nm. The standard solution was prepared by mixing 1 mL of the stock standard solutions of synapic acid, myricetin, vanillic acid, quercetin, gallic acid, syringic acid, epicatechin, naringenin, p-coumaric acid, caffeic acid, and luteolin, and we injected in triplicate (Table 1).
Plant extract (mg/kg)
Results are expressed as .