Anti-inflammatory effect of the Salvia sclarea L. ethanolic extract on lipopolysaccharide-induced periodontitis in rats

Author’s Accepted Manuscript Anti-inflammatory effect of the Salvia sclarea L. ethanolic extract on lipopolysaccharide-induced periodontitis in rats Milica Kostić, Dušanka Kitić, Milica B. Petrović, Tatjana Jevtović-Stojmenov, Marko Jović, Aleksandar Petrović, Slavoljub Živanović www.elsevier.com/locate/jep

PII: DOI: Reference:

S0378-8741(17)30169-1 http://dx.doi.org/10.1016/j.jep.2017.01.020 JEP10667

To appear in: Journal of Ethnopharmacology Received date: 20 June 2016 Revised date: 27 December 2016 Accepted date: 12 January 2017 Cite this article as: Milica Kostić, Dušanka Kitić, Milica B. Petrović, Tatjana Jevtović-Stojmenov, Marko Jović, Aleksandar Petrović and Slavoljub Živanović, Anti-inflammatory effect of the Salvia sclarea L. ethanolic extract on lipopolysaccharide-induced periodontitis in rats, Journal of Ethnopharmacology, http://dx.doi.org/10.1016/j.jep.2017.01.020 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Anti-inflammatory effect of the Salvia sclarea L. ethanolic extract on lipopolysaccharide-induced periodontitis in rats

Milica Kostića, Dušanka Kitića, Milica B. Petrovićb, Tatjana Jevtović-Stojmenovc, Marko Jovićd, Aleksandar Petrovićd, Slavoljub Živanovića a Department of Pharmacy, Faculty of Medicine, University of Niš, Blvd Dr Zorana Ðinđića 81, 18000 Niš, Serbia b Department of Stomatology, Faculty of Medicine, University of Niš, Blvd Dr Zorana Ðinđića 81, 18000 Niš, Serbia c Department of Biochemistry, Faculty of Medicine, University of Niš, Blvd Dr Zorana Ðinđića 81, 18000 Niš, Serbia d Department of Histology, Faculty of Medicine, University of Niš, Blvd Dr Zorana Ðinđića 81, 18000 Niš, Serbia

ABSTRACT Ethnopharmacological relevance Salvia sclarea L., clary, is an aromatic plant traditionally used in folk medicine for the treatment of various diseases and conditions. Although it has been primarily used as a stomachic, there are data on traditional use of S. sclarea as an agent against gingivitis, stomatitis and aphthae. Aim of the study: The aim of the study was to examine the effect of the S. sclarea ethanolic extract on the lipopolysaccharide (LPS)-induced periodontitis in rats from the immunological and histopathological standpoint. Material and methods Periodontal inflammation in rats was induced by repeated injections of LPS from Escherichia coli into the interdental papilla between the first and second right maxillary molars. The extract was administered two times a day by oral gavage (200 mg/kg body weight). The inflammatory status was assessed by the measurements of proinflammatory cytokines interleukin-1β (IL-1β),

interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) of gingival tissues and descriptive analysis of histological sections of periodontium. Chemical characterization of the extract was determined using high performance liquid chromatography system (HPLC). Antioxidant activity of the extract was estimated with two in vitro complementary methods: 2,2-diphenyl-1picrylhydrazyl and β-carotene/linoleic acid models. Results Treatment with S. sclarea extract, compared to the untreated group of the rats, significantly diminished the process of inflammation decreasing the levels of IL-1β, IL-6 and TNF-α, reducing the gingival tissue lesions and preserving bone alveolar resorption. Considerably smaller number of inflammatory cells and larger number of fibroblasts was noticed. The administration of the extract three days earlier did not have significant preventive effects. Rosmarinic acid was the predominant compound in the extract. The extract showed strong antioxidant effects in both test systems. Conclusions S. sclarea extract manifested anti-inflammatory effect in LPS-induced periodontitis suggesting that it may have a role as a therapeutic agent in periodontal diseases. Having in mind that overproduction of reactive oxygen species is connected to periodontitis, the strong antioxidant capacity may be contributable to anti-inflammatory properties of the extract.

Graphical abstract

Abbreviations

ANOVA, analysis of variance; BHA, butylated hydroxyanisole;

BHT, butylated

hydroxytoluene; DAD, diode array detector; DPPH, 2,2-diphenyl-1-picrylhydrazyl; ELISA, enzyme-linked immunosorbent assay; GAE, gallic acid equivalents; HPLC, high performance liquid chromatography; HE, hematoxylin and eosin; IC50, inhibitory concentration reducing 50% of radicals; IL-1β, interleukin-1β; IL-6, interleukin-6; LPS, lipopolysaccharide; PVPP, polyvinylpolypyrrolidone; ROS, reactive oxygen species; RANKL, receptor activator of nuclear factor kappa-B ligand; RA, rosmarinic acid; TNF-α, tumor necrosis factor-α; UV/VIS, ultraviolet–visible spectroscopy Keywords Salvia sclarea L., ethanolic extract, lipopolysaccharide-induced periodontitis, antioxidant activity, rosmarinic acid, rats

1. Introduction Periodontal disease is a chronic infective disease of the periodontium caused by periodontopathic bacteria accumulated on the tooth surface, characterized by destruction of the tooth supporting tissues including alveolar bone resorption (Bentzen et al., 2005; Dumitrescu et al., 2004). Subgingival Gram-negative organisms containing lipopolysaccharides (LPS) cause inflammation of the periodontal tissues inducing a local response, which implies a polymorphonuclear leukocyte infiltration, a production of reactive oxygen species (ROS) and inflammatory mediators such as cytokines and prostaglandins, amplification of lytic enzymes and activation of osteoclasts, oedema and vascular dilatation (Page, 1991; Kjeldsen et al., 1993;

Maruyama et al., 2011; Sculley and Langley-Evans, 2002). Recent investigations have shown that the gingival tissue from the inflamed area contains increased levels of some cytokines, primarily interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) (Irwin and Myrillas, 1998; Stashenko et al., 1991; Vahabi et al., 2011), implying that these cytokines mainly participate in the pathogenesis of periodontitis (Agarwal et al., 1995). The culminating stadium of damaging effects of the produced cytokines and ROS is destruction of tooth-supported tissues including connective and mineralized tissue (Maruyama et al., 2011; Rogers et al., 2007). A remarkable number of studies have demonstrated the use of plants to be a new trend in the prevention and treatment of periodontal diseases with less adverse effects on humans. Some drugs for oral inflammations treatment can change oral microbiota and cause teeth discoloration (Feres et al., 2015; Palombo, 2011; Sweetman, 2007). In addition, they can provoke gastrointestinal disturbances usually followed by diarrhea, nausea and vomiting, or rarely, cause phototoxicity and accumulation in bones and teeth (Sweetman, 2007). Therefore, the search for natural products or phytochemicals is growing increasingly with prospects to become the acceptable alternatives or complementary agents for oral inflammations (Palombo, 2011). Bioactive phytochemicals from herbs such as phenolic acids, flavonoids, tannins, terpenoids, alkaloids etc. have been found to be particularly helpful in periodontitis due to their potent antimicrobial and anti-inflammatory activities (Kumar et al., 2009). Plant species from the genus Salvia L., Lamiaceae family, especially Salvia officinalis L., are well-known effectual agents whose active components could reduce gingival inflammation, inhibit growth of plaques and have beneficial effects on the cavity prophylaxis (Ehrnhöfer-Ressler et al., 2013; Willershausen et al., 1991).

Salvia sclarea L. is a spicy, aromatic plant, known as anti-inflammatory, antimicrobial, antioxidant, antispasmodic, anticonvulsant, hypoglycemic and digestive agent (Moretti et al., 1997; Hammer et al., 1999; Miliauskas et al., 2004; Mantle et al., 2000; Leporatti et al., 1985). From herbal literatures, aqueous extracts of the plant have been used against various digestive disorders, as well as decoction and infusion for treating polyarthritis and acute rheumathism (Lawrence, 1994; Peana and Moretti, 2002; Rajagopal et al., 2013). Although there are data on traditional use of S. sclarea against gingivitis, stomatitis and aphthae (Leporatti et al., 1985), there have been no studies concerning its inhibiting impact on the development of periodontitis on an animal model; in addition this is the first experimental research focusing on antiinflammatory properties of the S. sclarea extract. In this pilot study, we investigated the effectiveness of the extract of S. sclarea in suppressing the inflammation of the rats periodontium caused by LPS, by determining the levels of proinflammatory factors and histopathological analysis, as well as the chemical composition and antioxidant activity of the same extract. 2. Material and methods 2.1. Chemical reagents All reagents and solvents in the investigations were of analytical or HPLC grade. Acetonitrile and methanol were purchased from J. T. Baker (Mallinckrodt Baker, Center Valley, PA) and chloroform from Carlo Erba Reagents S. A. S. (Val de Reuil, France). Ethanol was purchased from Zorka Pharma, Šabac, Serbia. Apigenin-7-O-glucoside, luteolin-7-O-glucoside and acacetin were obtained from Carl Roth (Karlsruhe, Germany) and butylated hydroxytoluene (BHT) from Supelco (Sigma Chemical Co., St Louis, MO, USA). Polyvinylpolypyrrolidone was obtained from Merck (Darmstadt, Germany). Caffeic acid and apigenin were purchased from Fluka

(Sigma Chemical Co., St Louis, MO, USA). Lipopolysaccharide, Folin-Ciocalteu reagent, trifluoracetic acid, gallic acid, 2,2-diphenyl-1-picrylhydrazyl, rosmarinic acid, butylated hydroxyanisole (BHA), β-carotene, linoleic acid, luteolin and sodium carbonate were purchased from Sigma Aldrich (Sigma Chemical Co., St Louis, MO, USA). 2.2. Plant material Aerial parts of S. sclarea, Lamiaceae family, in full flowering stage, were collected in the surrounding area of Nis, Serbia (GPS Coordinates: 43°33’05.27” N, 22°03’06.26” E; cca 334 m a.s.l.). Plant material was identified by Dr. Bojan Zlatkovic (Department of Biology and Ecology, Faculty of Science and Mathematics, University of Nis) and a voucher specimen has been deposited in the Herbarium of the Institute of Botany and Botanical Garden “Jevremovac”, University of Belgrade, Serbia (No. 17077). 2.3. Plant extraction The plant material was primarily air-dried, protected from direct effect of sunlight and then milled to powder. The powdered material was extracted three times with 80% (v/v) ethanol (1:10) in an ultrasonic bath for 20 minutes. Final extract was obtained after filtration through Watman paper and total removal of the solvent in a rotary vacuum evaporator at 40°C. The average yield of extraction was 15.31 ± 1.73%. Prior to the experiment, the solid extract was kept in a sealed bottle in a dark place at 4°C. 2.4. Experimental design with animals All experimental procedures were approved by the Ethical committee of Medical faculty, University of Nis (No 01-9002-03) and were performed in accordance with Good Scientific

Practice. Thirty male Wistar rats, obtained from vivarium of Medical faculty in Nis, ten weeks old (247.5 ± 54.16 g), were kept in wire cages, in a room with proper, controlled conditions of temperature (21-23°C) and humidity (65–70%) with 12 hours light/dark cycle. The animals were fed with milled pellets, and had free access to water during the experiment. Rats were anaesthetized

with

a

10%

ketamin-hydrochloride

solution

(2

mL/kg)

administered

intraperitoneally. Lipopolyssacharide, derived from Escherchia coli (055:B5) and diluted in a sterile saline solution, was used for induction of periodontitis. One microliter of LPS solution (10 µg/µL) was slowly injected into interdental papilla between the first and second right maxillary molar (Dumitrescu et al., 2004), two times for ten days, using a Hamilton microsyringe. Rats were randomly divided into five experimental groups with six animals in each: (I) injected with saline, treated with vehicle (distilled water); (II) injected with saline, treated with S. sclarea extract; (III) injected with LPS, treated with vehicle; (IV) injected with LPS, treated with S. sclarea extract; (V) injected with LPS, treated with S. sclarea extract three days before LPS injection and during the period of examination. Groups I and II received the same volume (1 µL) of sterile saline injection in the same location of rat maxilla. Groups II, III, IV and V received S. sclarea extract dissolved in water which was administered two times daily by oral gavage (200 mg/kg body weight). Administration of dissolved extracts or vehicle started the same day when injections were given except V group which was treated with the extract preventively, three days before LPS injections. After ten days, the rats were sacrificed by an overdose of anesthetic ketamine, then the maxillae were isolated and hemisected. The soft tissues around the molars of rats were carefully anatomized and prepared for biochemical analysis. Some of the sections from each group

including molars with their encircling tissues were subjected to a standard histological procedure. 2.5. Histopathological analysis Histopathological analysis was performed at the Department of Histology and Embryology, Medical Faculty, University of Nis. The material, molars with the surrounding gingival tissues, was rinsed in saline and fixed in 10% formaldehyde solution for up to 7 days and then subjected to a process of demineralization, due to the presence of bone elements and teeth, for 15 days in a 18% solution of disodium ethylenediaminetetraacetate. After the completion of decalcination, the material was cut into thin parts in the mesial-distal direction and treated by conventional histological procedures to paraffin molds. The final preparations were 4 µm thick stained with hematoxylin and eosin (HE). 2.6. Measurements of the factors of inflammation (TNF-α, IL-1β, and IL-6) in rats’ gingival tissues The levels of the factors of inflammation, TNF-α, IL-1β, and IL-6, were determined using supernatants of 5% homogenate made up of fresh gingival tissues with deionized water. The measurement of cytokines was performed by enzyme-linked immunosorbent assay (ELISA) kits specific for rats in accordance with the instructions provided by the manufacturer (Invitrogen, Life Technologies, USA). Briefly, 50 µL of sample was added to respective wells of a plate followed by 50 µL of a standard diluent buffer, and 50 µL of incubation buffer for IL-6 assay. After incubation and washing of the wells 100 µL of appropriate biotin conjugate was added, then the plates were left to incubate again followed by washing. Next stage included adding of 100 µL and incubation with streptavidin-HRP working solution. After this step and washing, the

plates were incubated with 100 µL of stabilized chromogen followed by final adding of 100 µL of stop solution. Absorbance of each well was read at 450 nm on ELISA microplate reader (Multiskan Ascent No354, Thermo Labsystems, Finland). A value of the sample concentration was calculated from a standard curve and twice multiplied. The results are presented as pg of the cytokines in mg of fresh gingival tissue. 2.7. Chemical composition of S. sclarea extract HPLC analysis was performed according to a method reported by Kostic et al. (2015) with certain modifications. Quantifications of the compounds were performed using Agilent 1200 HPLC system (Agilent Technologies, Palo Alto, Calif., U.S.A.) with a diode array detector (DAD). The analytical column was Purospher STAR RP-18e (150 × 4.6 mm) with particle size of 5 μm, manufactured by Merck, Darmstadt, Germany. The extract was dissolved in deionized water to a concentration of 10 mg/mL. Injected volume of the extract was 10 μL and column flow was 0.7 mL/min. The eluent system consisted of 0.1% water-trifluoroacetic acid solution (A) and acetonitrile (B) with linear gradient: 0-3 min 5-5% B, 3-32 min 5-28% B, 32-44 min 2550% B, 44-52 min 50-80% B, 52-54 min 80-90% B, 54-59 min 90-5% B, and 59-60 min 5% B. Operating temperature was maintained at 30ºC. The compounds were quantified on the basis of UV-VIS signal response in comparison to standards using calibration curves. The results are expressed as µg of compounds in mg of the extract. 2.8. Determination of total polyphenols and tannins The content of total phenolic compounds was determined by the colorimetric, Folin-Ciocalteu method. 0.02 mL of extract, dissolved in concentrated ethanol, was transferred into the test tubes followed by 0.25 mL of Folin-Ciocalteu reagent, previously diluted with

distilled water 1:1 (v/v), and 1.25 mL of 86.6 mg/mL Na2CO3 solution. Absorbance was measured at 725 nm on UV/VIS spectrophotometer (Evolution 60 Thermo scientific spectrophotometer, Fisher Scientific, Loughborough, U.K.) against a blank which contained solvent instead of extract solution, after vigorous shaking, 40 min after blue colour development. Quantification was calculated according to a standard curve made up with gallic acid. The result is expressed as mg of gallic acid equivalents (GAE) per gram of the extract. Total tannins content was determined by the same Folin-Ciocalteu method which implied primarily the removal of tannins by adsorption on an insoluble binding matrix (polyvinylpolypyrrolidone, PVPP). The assay was performed with clear supernatants and the results were expressed as mg GAE per gram of the sample in accordance with the standard calibration curve (FAO, IAEA, 2000). 2.9. 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay The ability of the extract to scavenge free-radicals was evaluated by DPPH method reported by Chang et al. (2010) with certain modifications. The extract was dissolved with ethanol in different decreasing concentrations. 40 μL of the solution were added to 120 μL of ethanol in the microtitre plate’s well followed by 40 μL of ethanolic DPPH solution (0.2 mg/mL). The plate was shaken for short and left in a dark place for half an hour. The absorbances of mixtures were measured by ELISA reader (Multiskan Ascent No354, Thermo Labsystems, Finland) at 550 nm. The percentage of free-radical inhibition was calculated using the equation: % inhibition = [(AK-AA)/(AK-AS)]×100

where AA is the absorbance of the extract solution, AK is the absorbance of the control (which consisted of the solvent and DPPH solution) and AS is the absorbance of the solvent. The concentration of the extract that inhibited 50% of DPPH radicals (IC50) was calculated from a curve which represented the dependence of the percentage of inhibition and the concentration of diluted extracts. Rosmarinic acid, BHT and BHA were used as positive controls. 2.10. Antioxidant assessment using the β-carotene/linoleic acid method The evaluation of the lipid peroxidation inhibitory activity was performed according to the β-carotene/linoleic acid method described by Koleva et al. (2002) with certain modifications. 2 mL of a solution, which was previously prepared by dissolving 2 mg of crystalline β-carotene in 10 mL of chloroform (HPLC purity), were pipetted into a round-bottom flask followed by 360 mg of Tween 20 and 50 μL of linoleic acid. After the total removal of chloroform under pressure in a rotary vacuum evaporator, 100 mL of oxidized water were added into the flask with shaking. This aqueous emulsion was pipetted, in aliquots of 200 μL, into the wells of 96-well microtitre plates containing 25 μL of serial ethanolic dilutions of S. sclarea extract. At the same time a solution deprived of β-carotene was prepared as a blank. The microtitre plates were gently shaken before the measuring of the zero time absorbance (A0) by ELISA reader (Multiskan Ascent No354, Thermo Labsystems, Finland) at 450 nm. Plates were placed in an incubator at 55°C, heated for 2 hours and then absorbances were measured again (A120). The percentage of inhibition of lipoperoxidation was calculated using the following equation (Barros et al., 2007): % inhibition = [(A120/ A0)]×100

The concentration of the extract that protected 50% of β-carotene (IC50) was calculated from concentration/% inhibition curve. Rosmarinic acid, BHT and BHA were used as the positive controls. 2.11. Statistical analysis All experimental results are presented as mean values of three parallel measurements plus or minus the standard deviations. The results were analysed by one-way analysis of variance (ANOVA) while differences among means were compared through Duncan test at p<0.05, considered as significant. Statistical analyses were carried out using SPSS 17 (SPSS, Inc., Chicago, IL).

3. Results 3.1. Effects of S. sclarea extract on histopathological characteristics of periodontitis in rats Morphological changes in HE preparations are not visible in the group I of rats. Figure 1A displays a cross section of rat molar with preserved structure of the tooth pulp tissue and periodontal ligament with a slight increase in the number of neutrophils. There are no perceptible changes in the alveolar bone. The histological characteristics of the HE slides of the experimental group II correspond completely to healthy tissue properties (Figure 1B). Compared to the control animals (I and II group) the histopathology of the animals subjected to periodontitis receiving the vehicle (III group) displays notable changes at HE slides. The changes are characterized by the expansion of blood vessels in the parodontium tissue and gingival increase in the number of neutrophils and lymphocytes. Inflammation is followed by resorption of alveolar bone with the visible osteoclasts on the surface of bone and increased bone marrow

space, strong deepening of the gingival sulcus and its separation from the cement teeth in the direction of the tooth root. The presence of red blood cells out of blood vessels indicating the bleeding (Figure 1C) is also evident. In the experimental groups IV and V (Figure 1D, 1E), which were subjected to S. sclarea extract and LPS, it can be observed much smaller number of inflammatory cells in regard to the third group of rats. In parodontium and gingival tissue a larger number of fibroblasts persist compared to other groups of experimental animals, while the gingival sulcus is in the normal position. The signs of bone resorption are not noticed.

A

B

C

D E I Fig. 1. Histopathological aspect of the effect of Salvia sclarea L. ethanolic extract on lipopolysaccharide (LPS)-induced periodontitis in rats. Sections with thickness of 4 µm were processed standardly and stained with hematoxylin and eosin. I group of rats were injected with

saline and treated with distilled water (A); II group - injected with saline, treated with Salvia sclarea L. extract (B); III group - injected with LPS, treated with vehicle (C); IV group - injected with LPS, treated with the extract (D); V group - injected with LPS, treated with the extract three days before LPS injection and during the period of examination (E). Morphological changes in Figure 1A and 1B are not visible. Figure 1C presents notable changes characterized by the development of inflammation and resorption of alveolar bone. Figure 1D and 1E displays the reducing of inflammatory process while the signs of bone resorption are not noticed.

3.2. Effects of S. sclarea extract on the gingival concentrations of TNF-α, IL-1β and IL-6 of rats The per os treatment with S. sclarea extract (IV and V group) significantly (p<0.05) decreased the level of TNF-α, IL-6 and IL-1β in the gingival tissue of rats with periodontitis when compared to the untreated group (III). There was no statistically significant difference of the levels of cytokines between groups which did not receive LPS (I and II). Preventive administration of the extract (V group) significantly reduced the levels of IL-1β (p<0.05), but not IL-6 and TNF-α, in comparison to the group whose treatment started the same day when the LPS was injected (IV).

pg of IL-6 in mg of gingival tissue

pg of IL-1β in mg of gingival tissue 2.5

A

2

1.5

1

0.5

0

0

a

I

a b c d

II III groups IV V

8

7

B

6

5

4

3

2

1

a a c b b

I II III IV V

groups

pg of TNFα in mg of gingival tissue

0.6

C 0.5 0.4 0.3 0.2 0.1 0

a

a

b

a,c

c

I

II

III

IV

V

groups

Fig. 2. The levels of the cytokines IL-1β (A), IL-6 (B) and TNF-α (C) in the gingival tissues of the rats. Rats from I group were injected with saline and treated with distilled water; II group injected with saline, treated with Salvia sclarea L. extract; III group - injected with LPS, treated with vehicle; IV group - injected with LPS, treated with the extract; V group - injected with LPS, treated with the extract three days before LPS injection and during the period of examination. The extract significantly decreased the levels of the cytokines (IV and V group) compared to untreated group of animals (III) (p<0.05). Values represent means ± standard deviation (N=6). Bars indicate the mean values and vertical lines standard deviations. Different letters in the columns show significant differences among varieties (p<0.05, Duncan test).

3.3.Chemical composition of S. sclarea extract and total polyphenolics and tannins content HPLC analysis of the chemical composition of the ethanolic extract from the aerial part of S. sclarea revealed the presence of the compounds displayed in Table 1. The sample was

scanned through next wavelengths: 220, 280, 330 and 360 nm. Figure 3. presents HPLCchromatogram recorded at 330 nm. The most dominant compound was rosmarinic acid. The amount of total polyphenolics was 129.87 ± 3.25 mg GAE/g, and tannins were present in quantity of 33.07 ± 1.07 mg GAE/g.

Table 1. HPLC characterization of the ethanolic extract from the aerial part of Salvia sclarea (μg of component in mg of extract) Compound Phenolic acids rosmarinic acid caffeic acid Flavonoid aglycones luteolin apigenin Flavonoid glycosides luteolin-7-O-glucoside apigenin-7-O-glucoside

μg/mg 165.30 ± 0.60 0.95 ± 0.03 0.50 ± 0.02 0.22 ± 0.01 5.55 ± 0.46 8.51 ± 0.80

Fig. 3. HPLC-chromatogram of the ethanolic extract of Salvia sclarea L. at 330 nm dissolved in water. Peaks: A – caffeic acid, B – a derivate of luteolin, C – luteolin-7-O-glucoside, D – apigenin-7-O-glucoside, E – rosmarinic acid, F – a derivate of apigenin, G – luteolin and H – apigenin

3.4. Antioxidant activity The extract demonstrated powerful antioxidant activity in both test systems. The effects were compared with rosmarinic acid and standard reference controls (positive controls) BHT and BHA. The results are presented in Table 2.

Table 2. Antioxidant activity of Salvia sclarea ethanolic extract and positive controls assessed by 2,2-diphenyl-1-picrylhydrazyl (DPPH) and β-carotene/linoleic acid models

extract RA BHT BHA

DPPH IC50 (µg/ml) 27.82 ± 2.51a 6.09 ± 0.48b 22.82 ± 2.07c 2.44 ± 0.09d

β-carotene assay IC50 (µg/ml) 19.13 ± 1.70a 32.55 ± 1.62b 0.03 ± 0.00c 0.04 ± 0.01c

Values represent means of three measurements ± standard deviation. Different superscripts column-wise are significant among varieties (p <0.05, Duncan test). RA = rosmarinic acid, BHT = butylated hydroxytoluene, BHA = butylated hydroxyanisole

4. Discussion Wistar rats were chosen to be used in the study as a model for periodontal disease since it has been shown that periodontal anatomy of molar region in rats are similar to that in humans. It has been proven that both, topical application, as well as the injection of LPS in mouse or rat periodontal tissue and gingiva develops common inflammatory changes, such as junctional epithelial disruption, infiltration of leukocytes and oedema of the subepithelial connective tissue (Dumitrescu et al., 2004.; Ijuhin et al., 1992; Miyauchi et al., 1998). Additionally, LPS injection in rats periodontal tissue and gingiva shows significant alveolar bone loss starting at second day after periodontitis induction, reaching a top at seventh day (Dumitrescu et al., 2006). Our histopathological findings in periodontal tissue and gingiva, obtained from the third group of

experimental animals, show similar changes in these tissues. Intragingival injection of 1 µL of LPS solution, twice for ten days, in interdental papilla between the first and second molars induced inflammatory responses which implied drastic changes in histological appearance, activation of immune cells and producing of proinflammatory cytokines in periodontal tissue (III group), unlike earlier findings that gingival LPS injection could not induce experimental periodontal disease (Bentzen et al., 2005). According to Dumitrescu et al. (2004) a total volume of LPS injection should be reduced to 1 µL since the firmly bound tissue of gingivae cannot accommodate and spread to an injection of 10 µL and much of injected solution could be spilled along needle track. Bacterial stimulation, especially by LPS, has been described to increase the secretion of proinflammatory cytokines by gingival inflammatory cells, especially key cytokines such as TNF-α, IL-1β and IL-6 (Vahabi et al., 2011). These cytokines play a relevant role in the initiation and development of periodontitis amplifying the immune response and activating of immune and nonimmune cells (Vahabi et al., 2011; Agarwal et al., 1995). They are also able to provoke remarkable tissue damage by induction of collagenase in fibroblasts and osteoclasts activation (Agarwal et al., 1995; Howells, 1995). The synergism between IL-1β and TNF-α and between IL-6 and TNF-α have been found, whereby TNF-α is likely more important cytokine (Vahabi et al., 2011). It has also been noticed that healthy gingival tissue may contain inflammatory cytokines including the ones mentioned above present in low amounts (Jandinski et al., 1991). This implies that cytokines are actually considerable factors for the maintenance of normal tissue homeostasis (Ejeil et al., 2003). Previous report about anti-inflammatory effects of S. sclarea was related to activity of its essential oil which conspicuously reduced inflammatory process in mice induced by carrageenin and histamine (Morreti et al., 1997). Our study has

shown that S. sclarea extract manifests anti-inflammatory effects in rats by significantly decreasing the levels of pro-inflammatory cytokines IL-1β, IL-6 and TNF-α (p<0.05) (Fig. 2) with visible histological changes. It is evident that the administration of the extract in IV and V groups of rats modulated host response with extensive reduction of inflammatory infiltrate in comparison to III group. Immunoassay revealed that there were not statistically significant differences in the levels of the factors of inflammation IL-6 and TNF-α in IV and V groups of the rats. Generally, there were no differences in histopathological characteristics between these groups suggesting no effects of the three days preventive administration of the extract. The control groups (I and II) were injected with saline solution, instead of LPS, in order to investigate possible effects of the injection on the initiation of inflammation and bone destruction. The histopathology of the periodontium of group I, which corresponded to the healthy tissue, showed similar appearance to the group II implying there were no visible traumatic effects of the needle, as it was also shown by immunoassay where there was no statistically difference in the level of cytokines between these groups. Having in mind that periodontitis is a very common condition in humans, there is an ongoing search of adequate preventive or therapeutic procedures. It has been shown that some substances, such as catechin from the green tea, have protective effects on the periodontal tissue and alveolar bone, when administered with LPS. It is believed that this effect is the result of immunomodulatory properties of catechins due to their ability to inhibit the osteoclastic bone resorption and osteoclast formation in the presence of RANKL (receptor activator of nuclear factor kappa-B ligand) both in vivo and in vitro (Nakamura et al., 2010) and by decreasing gingival oxidative stress and expression of proinflammatory cytokines (Maruyama et al., 2011). Curcumin, a dietary spice, and its chemically modified form, triketonic phenylamino carbonyl

curcumin, were also shown to produce a significant reduction of the inflammatory mediators in LPS induced periodontitis (Guimaraes et al., 2012; Elburki et al., 2014). Curcumin increased collagen content and fibroblastic cell numbers and it is believed that it potently inhibits innate immune responses associated with periodontal disease (Guimaraes et al., 2012). Baicalin, a type of flavonoid, can inhibit tissue’s inflammatory response in LPS induced periodontal disease, probably through decreasing the release of TNF-α, IL-1β, IL-6 and other factors, which had been confirmed that might enhance osteoclast function and promote bone resorption directly or indirectly (Chen et al., 2008). Total polyphenolic content in the extract was 129.87 ± 3.25 mg GAE/g which is much higher compared to majority of S. sclarea extracts from other studies (14.83 ± 0.80 mg GAE/g – 97.84 ± 1.17 mg GAE/g) (Derakhshani et al., 2012; Dudonne et al., 2009; Firuzu et al., 2013; Tulukcu et al., 2009; Gulcin et al., 2004; Tusevski et al., 2014; Miliauskas et al., 2004). Methanolic and aqueous extracts from Greek S. sclarea contained greater amounts of polyphenolics (169 and 242 mg GAE/g, respectively) (Stagos et al., 2012), as well as methanolic extracts from Iran (268 ± 8 mg GAE/g) (Asadi et al., 2010). HPLC-DAD method revealed the presence of phenolic acids, rosmarinic and caffeic, and flavonoids (Fig. 3). The predominant polyphenol in the extract was rosmarinic acid with the concentration of 165.30 ± 0.60 μg/mg. Previous investigations of its content in S. sclarea extracts showed significantly lower concentrations, 3.9 mg/g (Janicsak et al., 1999) and 41.1 mg/g (Bandoniene et al., 2005) in methanolic leaves extracts. The caffeic acid content is considerably higher in our extract compared to the methanolic leaf extract, as well, with the value of 0.08 mg/g (Janicsak et al., 1999). Luteolin, apigenin and their heterosides were only identified flavonoids. Due to the lack of standards, compounds B and F (Fig. 3) could only be recognized, on the basis of UV/VIS

spectra as a derivate of luteolin (B) and a derivate of apigenin (F). Beside apigenin and luteolin, Ulubelen et al. (1994) identified 4’-methylapigenin, 6-hydroxyluteolin-6,7,3’,4’-tetramethyl ether and 6-hydroxy apigenin-7,4’-dimethyl ether in acetone extract of the whole S. sclarea plant. A large number of pharmacological effects are attributed to the polyphenols, which often come from their antioxidant activity (Scalbert et al., 2005). Previous investigations showed that aqueous, methanolic and petroleum ether extracts of S. sclarea manifested potent antiradical activity (Orhan et al., 2007; Stagos et al., 2012; Tepe et al., 2006). Our S. sclarea extract showed significant ability to scavenge free radicals in DPPH system with IC50 = 27.82 ± 2.51 µg/mL, as well and its antilipoperoxidant activity in β-carotene/linoleic acid model was also considerable with the value of IC50 = 19.13 ± 1.70 µg/mL. The strong antioxidant capacity may contribute to its anti-inflammatory properties in the periodontal disease. It has been evident that overproduction of ROS is connected to periodontitis. The treatment with Angipars, a drug derived from Melilotus officinalis, significantly decreased the levels of the markers of the oxidative damages in rats’ gingiva (Mousavi-Jazi et al., 2010). Rosmarinic acid was proven to be an excellent antiradical antioxidant with IC50 = 6.09 ± 0.48 µg/mL, but on the other hand it was less active in lipoperoxidation-inhibitory activity. The standards BHT and BHA were potent in both test systems, especially in β-carotene/linoleic acid model, as expected. In addition to the outstanding antiradical activity, rosmarinic acid manifests considerable anti-inflammatory effects (Petersen and Simmonds, 2003). Significant quantity of rosmarinic acid (165.30 ± 0.60 μg/mg) and literature data imply that the effects of S. sclarea extract may be primarily attributed to this compound. Namely, this phytochemical reduced the depletion of glutathione, production of ROS, lipid peroxidation and expression of IL-1β, IL-6, TNF-α in human gingival fibroblasts

treated with LPS (Zdarilova et al., 2009). Although the contents of apigenin, luteolin and caffeic acid are lower, their impact on the extract’s effectiveness should not be disregarded. Apigenin and luteolin were efficient in the inhibition of the process of proliferation of dental pulp cells and human periodontal ligament cells with the increasing of apoptosis (Liu et al., 2015; Liu et al., 2016). Gutiérrez-Venegas et al. (2006) established that luteolin pretreatment in human gingival fibroblasts subjected to LPS blocked nitric oxide synthesis and cellular activation of some mitogen-activated protein kinase family members, and repealed nuclear factor kappa B translocation. Jeong et al. (2009) found that apigenin was able to reduce the LPS-induced production of nitric oxide, prostaglandin E2, IL-1β, TNF-α, IL-6 and IL-12 in human periodontal ligament cells, as well as the upregulation of cyclooxigenase-2 and inducible nitric oxide synthase. They also revealed that apigenin expressed anti-inflammatory activity by a novel mechanism, which included the involvement of heme oxygenase-1. Although there is a lack of information about anti-inflammatory effectiveness of caffeic acid from the stomatological aspect, it was shown that this phytochemical can low levels of cytokines and production of nitric oxide (Ogiwara et al., 2003; Miles et al., 2005). The values of IL-1β were considerably decreased by 40% in the presence of caffeic acid in LPS-inflamed mononuclear human cells, but it could not inhibit the production of TNF-α, IL-6 and prostaglandin E2 (Miles et al., 2005).

5. Conclusion This study demonstrated that the oral administration of the ethanolic extract from S. sclarea had a suppressive effect on the LPS-induced inflammation of the rats’ gingival tissue. The decrease of cytokines production IL-6, IL-1β and TNF-α was in correlation with the reduction of the inflammatory process confirmed by histopathological analysis. The extract acted as a strong antioxidant agent, predominantly as the scavenger of free radicals, so that its role in

periodontitis may derive from antioxidant characteristics. The extract contained relevant quantity of polyphenols primarily including high level of rosmarinic acid which probably mostly contributed to these pharmacological effects. In general, the results suggest that S. sclarea could act as a potential therapeutic and complementary agent in the treatment of periodontal disease. However, further studies should be conducted in order to confirm and define the efficiency in humans in certain clinical researches.

Acknowledgment This study was financially supported by the Ministry of Science and Technological Development of Republic of Serbia, Grant Nos. III 41018 and III 46013, and the NIH/Fogarty International Center USA, Grant D43 TW00641 Training and Research in Environmental Health in the Balkans. The authors are also grateful for the financial support of the Internal project of Faculty of Medicine, University of Nis, named "Chemical characterization, biological activity and nutritional value of Ribes nigrum L, Salvia sclarea L. and Foeniculum vulgare Miller". The authors are very thankful to Dr Bojan Zlatkovic (Department of Biology and Ecology, Faculty of Science and Mathematics, University of Nis) for identification of the plant material and kindly providing of accession number in the Herbarium of the Institute of Botany and Botanical Garden “Jevremovac”, University of Belgrade, Serbia.

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Table 1. HPLC characterization of the ethanolic extract from the aerial part of Salvia sclarea (μg of component in mg of extract) Compound Phenolic acids rosmarinic acid caffeic acid Flavonoid aglycones luteolin apigenin Flavonoid glycosides luteolin-7-O-glucoside apigenin-7-O-glucoside

μg/mg 165.30 ± 0.60 0.95 ± 0.03 0.50 ± 0.02 0.22 ± 0.01 5.55 ± 0.46 8.51 ± 0.80

Table 2. Antioxidant activity of Salvia sclarea ethanolic extract and positive controls assessed by 2,2-diphenyl-1-picrylhydrazyl (DPPH) and β-carotene/linoleic acid models

extract RA BHT BHA

DPPH IC50 (µg/ml) 27.82 ± 2.51a 6.09 ± 0.48b 22.82 ± 2.07c 2.44 ± 0.09d

β-carotene assay IC50 (µg/ml) 19.13 ± 1.70a 32.55 ± 1.62b 0.03 ± 0.00c 0.04 ± 0.01c

Values represent means of three measurements ± standard deviation. Different superscripts column-wise are significant among varieties (p <0.05, Duncan test). RA = rosmarinic acid, BHT = butylated hydroxytoluene, BHA = butylated hydroxyanisole

LPS-induced periodontitis

*Graphical Abstract OH OH

O O

O

OH

OH OH

IL-6

TNFIL-1!