Modulation of the Inflammatory Response in Murine Macrophages by Mastic Essential Oil

MODULATION OF THE INFLAMMATORY RESPONSE IN MURINE MACROPHAGES BY MASTIC ESSENTIAL OIL

Georgia A. Kolezaki

Supervisor: Dr. K. Chlichlia

Alexandroupolis, 2016

CONTENTS

Acknowledgements …………………………………………………...... 6

Abstract ……………………………………………………………………………………………………….. 7

1.Introduction ……………………………………………………………………………………………… 8

1.1 Inflammation …………………………………………………………………………………………….. 8

1.1.1Definition ………………………………………………………………………………………………… 8

1.1.2 Inflammatory Mediators …………………………………………………………………………. 9

1.1.2.1 Nitrite Oxide (NO) ……………………………………………………………………………….. 9

1.1.2.2 Tumor Necrosis Factor-α (ΤΝF-α)………………………………………………………….10

1.1.2.3 PGE2………………………………………………………………………………………………………11

1.1.3 LPS-induced inflammatory response………………………………………………………..13

1.1.4 Phagocytosis and the inflammatory response………………………………………….14

1.2 Pistacia lentiscus var. Chia…………………………………………………………………………..17

1.2.1 General Characteristics…………………………………………………………………………….17

1.2.2 Taxonomy………………………………………………………………………………………………..18

1.2.3 Mastic Oil…………………………………………………………………………………………………19

1.2.3.1 Essential Oils …………………………………………………………………………………………19

1.2.3.2 Chemical Composition…………………………………………………………………………..20

1.2.4 Biological Activities of mastic tree oil and resin………………………………………..20

1.2.4.1 Anti-cancer Activity……………………………………………………………………………….20

1.2.4.2 Anti-oxidant Activity……………………………………………………………………………..21

1.2.4.3 Antibacterial Activity…………………………………………………………………………….21

1.3 Anti-inflammatory properties of mastic oil’s major components………………..22

1.4 Aim……………………………………………………………………………………………………………..23

2. Materials and Methods………………………………………………………………………………24

2.1 Mastic oil’s major components…………………………………………………………………..24

2.2 Cell Cultures………………………………………………………………………………………………..27

2.3 SRB Assay…………………………………………………………………………………………………….29

2.4 Griess Test…………………………………………………………………………………………………..30

2.5 Enzyme Linked Immunosorbent Assay (ELISA)……………………………………………..31

2.6 Prostaglandin E2 (PGE2) Assay………………………………………………………………………33

2.7 Comet Assay………………………………………………………………………………………………..35

2.8 Phagocytosis Assay………………………………………………………………………………………38

2.9 Fluorescence Microscopy……………………………………………………………………………40

2.10 Optical Microscopy…………………………………………………………………………………….41

2.11 Statistical Analysis……………………………………………………………………………………..41

3. Results………………………………………………………………………………………………………..42

3.1 Determinaton of the maximum non toxic concentrations of mastic oil and its major components…………………………………………………………………………………………….42

3.2 Mastic and b-pinene reduce significantly the production of NO, PGE2 and ΤΝF-α in supernatants of LPS-induced RAW264.7 cells………………………………………………..43

3.3 Mastic, limonene and linalol reduce the %DNA-damage caused by LPS in RAW264.7 cells………………………………………………………………………………………………….46

3.4 Inhibition of Morphological Changes in LPS-stimulated RAW264.7 Macrophages by mastic oil………………………………………………………………………………………………………45

3.5 Limonene, linalol and mastic oil reduce the rate of phagocytosis of L. casei by RAW 264.7 cells………………………………………………………………………………………………..49

4. Discussion…………………………………………………………………………………………………..52

5. References………………………………………………………………………………………………….55

Acknowledgements

This study was conducted for my thesis as a requirement for the completion of my undergraduate studies in Molecular Biology and Genetics.

First of all, I would like to thank my supervisor Dr. K. Chlichlia for her continued support, interest and time that she dedicated in order this study to be carried out. I would also like to express my gratitude to the PhD student K. Spyridopoulou for her help and useful instructions either during the laboratory work or the writing of this dissertation. Also, I would like to thank all the laboratory team for their help and support as well as Dr. A. Pappa and the PhD student E. Fitsiou for their help in carrying out the Comet assay experiments.

Abstract

Pistacia lentiscus var. Chia (mastic tree) is a member of the species Pistacia lentiscus that is grown in Chios island in Greece. The mastic resin is produced from the bark of the trees and, although it is considered to have significant biological activities such as antimicrobial, antioxidant and anticancer and has been used in pharmaceutical products and nutritional supplements, little is known about its role in modulating the inflammatory response. In this study the modulation of the inflammatory response by the essential oil of Chios mastic resin and its major constituents was comparatively evaluated using an in vitro inflammation model based on LPS-induced murine macrophages RAW264.7. After the determination of the non-toxic concentrations of mastic oil or its major components (b-pinene, α-pinene, myrcene, linalol and limonene) to RAW264.7 cells, their anti-inflammatory potency was investigated by assaying their effects on inflammation mediators production/secretion and inflammatory responses induced in RAW264.7 cells by LPS stimulation. Mastic oil and some of its bioactive constituents suppressed a) NO production, b) prostaglandin E2 secretion and c) TNF-alpha production in LPS- stimulated RAW264.7 cells. Moreover, the percentage of RAW264.7 cells that underwent LPS-induced morphological changes was smaller after pre-tretment of the the cells with either mastic or some of its constituents. Additionally, mastic oil, linalol and limonene were found to possess a protective role against DNA-damage caused by LPS. Finally, mastic oil and its major constituents reduced the rate of phagocytosis on murine macrophage. Thus, mastic oil and its bioactive components modulate the inflammatory response, suggesting that mastic oil promotes an anti- inflammatory transition in LPS-stimulated murine macrophages.

1. INTRODUCTION

1.1 Inflammation

1.1.1 Definition

Inflammation is part of the complex biological response of our body tissues to harmful stimuli such as pathogens or damaged cells. It is a protective response involving immune cells, blood vessels and molecular mediators. The role of inflammation is to eliminate the initial cause of injury, clear out necrotic cells and repair tissues [1]. The classical signs of acute inflammation are heat, pain redness, swelling and loss of function. Inflammation is a generic response and therefore it is considered as a mechanism of innate immunity. It can be classified as either acute or chronic. Acute inflammation is achieved by the increased movement of plasma and leukocytes from blood into tissues, whether chronic inflammation is characterized by simultaneous destruction and healing of the tissue [2].

Figure 1: Acute vs Chronic Inflammation. (Baniyash, Michal. "TCR ζ-chain downregulation: curtailing an excessive inflammatory immune response." Nature Reviews Immunology 4.9 (2004): 675-687.)

1.1.2 Inflammatory Mediators

1.1.2.1 Nitrite Oxide (NO)

An important mediator in acute and chronic inflammation is nitric oxide (NO). NO is generated via the oxidation of the terminal guanidino nitrogen atom of L-arginine by the enzyme, nitric oxide synthase (NOS). Three major isoforms of NOS have been identified. Two expressed constitutively, are calcium/calmodulin-dependent and are classified together as constitutive NOS isoforms (cNOS). The third is a cytokine- inducible, calcium/calmodulin-independent isoform of NOS (iNOS). NO has been shown to increase the production of pro-inflammatory prostaglandins in vitro, ex vivo and in vivo studies, potentially by S-nitrosation of cysteine residues in the catalytic domain of cyclo-oxygenase (COX) enzymes. In addition, NO can also react with superoxide anion to form peroxynitrite (ONOO-), a potent oxidizing molecule capable of eliciting lipid peroxidation and cellular damage. These findings suggest that NO has the ability to exert multiple cytotoxic effects during inflammatory responses including an increase PG production as well as the formation of ONOO- [3].

Figure 2: Nitrite Oxide (NO) synthesis reaction (Lauer T., Kleinbogard P., Kelm M. Indexes of NO Bioavailability in Human Blood. American Physiological Society, 2002, 17: 251-255).

1.1.2.2 Tumor Necrosis Factor-α (TNF-α)

TNF-α was discovered in 1975 as an endotoxin-inducible molecule that caused necrosis of tumors in vitro. It is a transmembrane 26kDa protein expressed by activated NK and T-cells, but also by a diverse array of non-immune cells such as endothelial cells and fibroblasts. The production of TNF-α mRNA is transcriptionally regulated by NF-κΒ, c-Jun, AP-1 and NFAT, consistent with the presence of these transcription factor binding sites within the promoter region of the TNF gene [4]. A plethora of in vitro studies have revealed complex and divergent TNF-R signaling pathways which account for all aspects of TNF’s ability to induce both cell death and/or co stimulation and cell activation [5].

TNFs’ stimulation of globally activating transcription factors such as NF-kB, and its signaling via bio-active lipids that induce arachidonic acid, 5-HETE and ultimately leukotrienes and prostaglandins, explain its effects on diverse cells within almost every human physiological system. They also explain TNFs powerful proinflammatory capacity, especially within immune cells capable of producing a cascade of downstream cytokines and chemokines [6]. For example TNF promotes monocyte/macrophage differentiation and enhances activated B cell proliferation concomitant with an autocrine increase in TNFR expression. It promotes the proliferation of fibroblasts and is a powerful inducer of inflammation, often acting together with IL-1b [7].

10 Figure 3: The TNF-α pathway. (Victor F.C., Gottlieb A.B., TNF-α and apoptosis implications for the pathogenesis and treatment of psoriasis. J. Drugs Dermatol., 2002, 1: 264-275). 1.1.2.3 Prostaglandin E2 (PGE2)

Prostaglandins are small-molecule derivatives of arachidonic acid (AA), produced by cyclooxygenases and PG synthases, with a relatively minor contribution of the isoprostane pathway. Local levels of PGE2, the main product of cyclooxygenases in myeloid and stromal cells, are regulated by the local balance between the COX2- driven synthesis and 15-hydroxyprostaglandin dehydrogenase (15-PGDH)-mediated degradation of PGE2. The receptors for PGE2 (EP1–EP4) are present on multiple cell types [8].

PGE2 has been shown to regulate multiple aspects of inflammation and multiple functions of different immune cells. Although generally recognized as a mediator of active inflammation, promoting local vasodilatation and local attraction and activation of neutrophils, macrophages and mast cells at early stages of inflammation, its ability to promote the induction of suppressive IL-10 and to directly suppress the proinflammatory cytokines allow it to limit non-specific inflammation, promoting the immune suppression associated with chronic inflammation and cancer [9]. Although PGE2 can promote the activation, maturation and migration of dendritic cells (the basic cells during the development of Ag-specific immunity) it has been widely demonstrating to suppress both innate and Ag-specific immunity at multiple molecular and cellular levels.

PGE2 can be produced by all cell type but the major sources of PGE2 are epithelia, fibroblasts and infiltrating inflammatory cells. The process of PGE2 synthesis involves phospholipase A2 family members that mobilize AA from cellular membranes, cycloxigenases that convert AA into PGH2 and PGE synthases needed for the final formulation of PGE2. Although the rate of PGE2 synthesis and the resulting inflammatory process can be affected by additional factors such as local availability of AA in most physiologic conditions the rate of PGE2 synthesis is controlled by local expression and activity of COX2 [10].

Figure 4: PGE2 synthesis. (Jungel A., Distler O., Schulze-Horsel U. Microparticles Stimulate the synthesis of prostaglandin E2 via induction of cyproxygenase 2 and microsomal prostaglandin E synthase 1. Arthritis & Rheumatology, 2007, 56: 3564-74).

1.1.3 LPS induced inflammatory response

LPS (Lipopolysaccharides) are large molecules consisting of a lipid and a polysaccharide composed of O-antigen, outer core and inner core joined by a covalent bond. They are found in the outer membrane of Gram-negative bacteria contributing to their structural integrity and protecting the membrane from certain kinds of chemical attack [11]. LPS also increases the negative charge of the cell membrane and helps stabilize the overall membrane structure. It is of crucial importance to Gram-negative bacteria, which die if it is mutated or removed. LPS induces a strong response from normal animal immune systems [12]. It has also been implicated in non-pathogenic aspects of bacterial ecology, including surface adhesion, bacteriophage sensitivity, and interactions with predators such as amoebae.

Figure 5: LPS Structure. (Serrato V.R. Lipopolysaccharides in diazotropic bacteria. Front. Cell Infect., Microbiol., 2014).

LPs acts as endotoxin because it binds the CD14/TLR4/MD2 receptor complex in many cell types, but especially in monocytes, dendritic cells, macrophages and B cells, that promote the secretion of pro-inflammatory cytokines, nitric oxide, and Prostaglandins [13]. Moreover, LPS is an exogenous pyrogen (external fever- inducing substance).

1.1.4 Phagocytosis and the Inflammatory ResponseCells have evolved a variety of strategies to internalize particles and solutes, including pinocytosis, receptor- mediated endocytosis, and phagocytosis. Phagocytosis, the uptake of large particles (>0.5 µm) into cells, occurs by an actin-dependent mechanism and is usually independent of clathrin.

Figure 6: Phagocytosis of a target particle. (D.M.Richards. The Mechanism of Phagocytosis: Two stages of Engulfment. Biophysical Journal, 2014, 107: 1542-1553).

While lower organisms use phagocytosis primarily for the acquisition of nutrients, phagocytosis in Metazoa occurs primarily in specialized phagocytic cells such as macrophages and neutrophils phagocytosis by macrophages is critical for the uptake and degradation of infectious agents and senescent cells [15]. It participates in development, tissue remodeling, the immune response, and inflammation. Monocytes/macrophages and neutrophils have been referred as professional phagocytes. On the other hand, most cells have some phagocytic capacity. For example, thyroid and bladder epithelial cells phagocytose erythrocytes in vivo, and numerous cell types have been induced to phagocytose particles in culture [16].

During inflammation many leukocytes are recruited through chemotaxis to the site of injury. These cells act as phagocytes ingesting bacteria, viruses, and cellular debris. Others release enzymatic granules that damage pathogenic invaders. Leukocytes also release inflammatory mediators that develop and maintain the inflammatory

14 response. In general, acute inflammation is mediated by granulocytes, whereas chronic inflammation is mediated by mononuclear cells such as monocytes and lymphocytes.

The extravasated neutrophils come into contact with microbes at the site of the inflamed tissue. Phagocytes express cell-surface endocytic pattern recognition receptors(PRRs) that have affinity and efficacy against non-specific microbe- associated molecular patterns (PAMPs). Most PAMPs that bind to endocytic PRRs and initiate phagocytosis are cell wall components such as lipopolysaccharides (LPS), including complex carbohydrates such as β-glucans, peptidoglycans, and surface proteins.

Upon endocytic PRR binding, actin-myosin cytoskeletal rearrangement adjacent to the plasma membrane occurs in a way that endocytoses the plasma membrane containing the PRR-PAMP complex, and the microbe. Phosphatidylinositol signalling pathways have been implicated to traffic the endocytosed phagosome to intracellular lysosomes, where fusion of the phagosome and the lysosome produces a phagolysosome. The reactive oxygen species, superoxides and hypochlorite bleach within the phagolysosomes then kill microbes inside the phagocyte.

Phagocytic efficacy can be also enhanced by opsonization. Plasma derived complement C3b and antibodies that exude into the inflamed tissue during the vascular phase bind and coat the microbial antigens. As well as endocytic PRRs, phagocytes also express opsonin receptors Fc receptor and complement receptor 1 (CR1), which bind to antibodies and C3b. The co-stimulation of endocytic PRR and opsonin receptor increases the efficacy of the phagocytic process, enhancing the lysosomal elimination of the infective agent.

Figure 7: FcγR-mediated Phagocytosis in Macrophages. (Nimmerjahn F., Ravetch J.V. Fcγ receptors as regulators of immune responses. Nature Innumol., 2008, 8:34-47).

1.2 Pistacia lentiscus var. Chia

1.2.1 General Characteristics

Pistacia lentiscus is an evergreen bush of the Anacardiacae family that extensively thrives in the eastern Mediterranean area of Europe. The variety chia is cultivated in the southern region of Chios Greek island and produces exclusively a white, semitransparent natural resin. The latter is known as Chios mastic gum and is obtained after hurting the trunk and the branches of the bush [19].

Knowledge of the benefits of mastic gum consumption goes back to ancient times, when ancient Greeks as Hippocrates and Galenos mentioned its properties. They recommended its use for the treatment of various gastrointestinal malfunctions such as gastralgia, peptic ulcer and dyspepsia [20]. In modern times mastic gum has been used as a supplement in liquors, drinks, foods, chewing gum, toothpaste, lotions and other cosmetics [21].

Figure 8: Mastic Resin

1.2.2 Taxonomy of Pistacia lentiscus var.chia

The plant Pistacia lentiscus var. chia also known as mastic tree is a member of the family Anacardiceae in which four genera are included:

 Rhus L.  Cotinus Miller  Schinus L.  Pistacia L.

The subspieces of genus Pistacia are the following:

1. Pistacia aethiopica Kokwaro 2. Pistacia atlantica Desf. 3. Pistacia chinensis Bunge 4. Pistacia eurycarpa Yalt 5. Pistacia integerimma J. Stewart 6. Pistacia khinjuk Stocks 7. Pistacia lentiscus L. 8. Pistacia mexicana Kunth 9. Pistacia terebinthus L. 10. Pistacia texana Swingle 11. Pistacia vera L. 12. Pistacia weinmannifolia J. Poiss ex Franch [22]

1.2.3 Mastic Oil

1.2.3.1 Essential Oils

Essential oils are volatile, natural, complex compounds formed by aromatic plants as secondary metabolites. They are usually obtained by steam or hydro-distillation first developed in the Middle Ages by Arabs. They are known for their antiseptic (bactericidal, virucidal, fungicidal) and pharmaceutical properties and they are used in antimicrobial, analgesic, sedative, anti-inflammatory, spasmolytic and locally anesthetic remedies [23].

In nature, essential oils play an important role in the protection of the plants as antibacterials, antivirals and anti-fungals. They also may attract some insects to favour the dispersion of pollens and seeds [24].

Essential oils are extracted from various aromatic plants generally localized in warm countries. They are rarely coloured They are soluble in organic solvents with a generally lower density than that of water. They can be synthesized by all plant organs. There are several methods for extracting essential oils. These may include use of liquid carbon dioxide or microwaves and mainly low or high pressure distillation.

Due to their bactericidal and fungicidal properties, their use in pharmaceutical and food industry as alternative to synthetic chemical products is getting more popular because it protects the ecological equilibrium. In those cases, extraction by steam distillation is preferred [25].

For perfume uses an extraction with lipophilic solvents and sometimes with carbon dioxide is preferred. Thus the chemical profile of the essential oil products differs not only in the number of molecules but also in the stereochemical types of molecules extracted depending on the type of extraction. The extraction product can vary in quality, quantity and in composition according to the climate, the soil composition, the plant’s organ and age. Most of the commercialized essential oils are chemotyped by gas chromatography and mass spectrometry analysis [26].

At present approximately 3000 essential oils are known, 300 of which are commercially important for the pharmaceutical, agronomic, food, sanitary, cosmetic and perfume industries.

Essential oils are very complex natural mixtures which can contain about 20-60 components at quite different concentrations. They are characterized by two or three major components and other constituents present in trace amounts [27].

1.2.3.2 Chemical Composition

The chemical composition of mastic oil has been analyzed and a few components have been isolated and identified in various fractions. Among them a-pinene, β- myrcene, β-pinene, limonene and β-caryophyllene are the major constituents of mastic essential oil. Several trace components such as verbenone, a-terpineol and linalol which appeared to contribute significantly to the antibacterial activity of mastic oil were also identified [28].

1.2.4 Biological Activities of mastic oil and resin

1.2.4.1 Anticancer Activity

Pistacia lentiscus gum has been traditionally regarded as an anti-cancer agent especially against of colon and uterus tumors [27]. These traditional beliefs are in agreement with recent studies demonstrating that Chios mastic induces apoptosis and possesses anti-proliferative activity [30].

In the last few years, a gradually increasing number of studies have evaluated the potential anti-proliferative properties of mastic oil against several types of human neoplasia setting a basis for its future application against cancer [17]. In the in vitro study by He ML. et al., it was shown that mastic gum (20-60 μg/ml) inhibits the proliferation and cell-cycle progression at G1 phase in prostate cancer PC-3 cells by suppressing NF-κΒ activity and NF-κΒ signal pathway [31].

In the study by Moulos P. et al., it was shown that treatment of Lewis lung carcinoma cells with mastic oil (0.01% v/v) led to a time dependent alteration in the expression of 952 genes [32]. Modifications on cell cycle proliferation, survival and NF- κΒ cascade indicated some important mechanistic links underlying the anti- proliferative, proapoptotic and anti-inflammatory effects of mastic oil [33]. Likewise, mastic oil (0.01-0.02% v/v) reduced vascular endothelial growth factor (VEGF) and chemokine release by Lewis lung carcinoma cells [34]. Furthermore, mastic oil administration decreased small guanosine thriphosphatases (GTPases), Ras, RhoA and NF-κΒ dependent reporter gene expression both in vivo and in vitro indicating a mechanistic link between mastic oil activities and blocking of relevant signaling and transcription pathways [36].

1.2.4.2 Antioxidant Activity

Mastic resin, as well as other resins, has been used for centuries as a preservative for fats and oils by people of various ethnicities [36]. The first study on mastic’s antioxidant activity showed that mastic possesses antioxidant activity similar to that of butylated hydroxyanisol [37]. In a more recent study where the potential protective effect of several resins upon the copper-induced oxidation of human low- density lipoprotein (LDL) was tested, the hydromethanolic extract of mastic gum was rated as the most active [38]. Several combinations of triterpenes including mastic oil compounds such as oleanolic acid showed considerable protective activity as well [39].

1.2.4.3 Antibacterial Activity

The first study mentioning anti-H.pylori activity, of mastic oil revealed the bactericidal activity of mastic upon H.pylori in vitro at concentrations as low as 60 μg/ml of broth culture. Lower concentrations were proven inhibitory for the growth of the bacterium and transmission electron spectroscopy revealed ultrastructural changes in the bacteria [40]. Studies that followed assessed the anti-bacterial activity of mastic upon isolated clinical strains of H.pylori in concentrations 1.9 to 2000 μg/ml. The calculation of the Minimum Bactericidal Concentrations showed that mastic exhibited considerable bactericidal effect upon the 12 H.pylori strains isolated

21 from patients that were used by killing 50% of the bacteria at a concentration of 125 μg/ml and 90% at a concentration of 500μg/ml [28].

After the discovery of anti-H.pylori effect of mastic followed studies regarding its general anti-microbial properties. It has been found that addition of mastic oil in broth cultures inoculated with Gram(+) (S.aureus and Lactobacillus plantarum) and Gram(-) bacteria (Pseudomonas fragi and Salmonella enteridis) inhibits their growth [40].

1.3 Anti-inflammatory properties of mastic oil’s major components

Some of the major components of mastic oil have been shown to possess anti- inflammatory activity. It was observed that limonene decreased the MCP-1 production in supernatants of Df-HL 60 clone 15 cells [42]. Limonene was shown to suppress the LPS-induced production of Nitrite Oxide (NO), Prostanglandin E2 (PGE2) and proinflammatory cytokines such as TNF-a, IL-6 and IL-1β in a dose dependent manner in RAW264.7 macrophages [43].

In the in vivo study by Peana et. al, linalol was shown to reduce NO production by LPS-stimulated macrophage cell line J774.A1 [54]. In another study by Huo et. al, it has been shown that LPS secretion of IL-6 and TNF-α is inhibited in a dose dependent manner after the treatment of Raw264.7 cells with linalol in a lung injury model [44].

Alpha-pinene (α-pinene) was found to exhibit anti-inflammatory activity through the suppression of mitogen-activated protein kinases (MAPKs) and the nuclear factor- kappa B (NF-κB) pathway in mouse peritoneal macrophages. Specifically, it decreased the LPS-induced production of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and nitric oxide (NO). α-pinene also inhibited inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expressions in LPS-stimulated macrophages [45]. Additionally, in an in vitro study by Rufino et. Al, a-pinene was found to elicite inhibition of IL-1β-induced inflammatory pathways namely NF-κΒ and JNK activation and the expression of iNOS [42].

1.4 Aim

The aim of this study was the investigation of the potential anti-inflammatory activity of mastic oil and its major components on an in vitro inflammation model based on LPS-treated RAW264.7 murine macrophages.

2.Materials and Methods

2.1 Mastic oil’s major components

In this study mastic oil and its major components were investigated for a potential anti-inflammatory activity. The phytochemical analysis of the essential mastic oil was determined through GC MS analysis (Table I).

compounds % area

α-pinene 67.71 camphene 0.70 verbenene 0.07

β-pinene 3.05 myrcene 18.81 limonene 0.89 linalol 0.73

α-campholenic ald 0.26 pinocarveol 0.32 trans-verbenol 0.07 cis-verbenol 0.69 verbenone 0.32 caryophyllene 0.50

The densities of mastic oil’s major components are shown in Table II.

Table ΙI: Densities of mastic oil constituents.

Mastic Oil/ Constituents Density (gr/ml) Mastic oil 0.89 limonene 0.87 linalol 0.89 myrcene 0.81 α-pinene 0.87 b-pinene 0.89

α-Pinene: is an organic compound of the terpene class, one of two isomers of pinene. It is an alkene and it contains a reactive four-membered ring. It is found in the oils of many species of many coniferous trees, notably the pine [46]. Both enantiomers are known in nature. (1S,5S)- or (−)-α-pinene is more common in European pines, whereas the (1R,5R)- or (+)-α-isomer is more common in North America. The racemic mixture is present in some oils such as eucalyptus oil and orange peel oil [47].

Figure 9: (+)-a-pinene structure.

beta-Pinene (β-pinene): is a monoterpene, an organic compound found in plants. It is one of the two isomers of pinene. It is colorless liquid soluble in alcohol, but not water. It has a woody-green pine-like smell [48].

Figure 10: beta-pinene structure.

Myrcene (or β-myrcene): is an olefinic natural organic hydrocarbon. It is more precisely classified as a monoterpene. Monoterpenes are dimers of isoprenoid precursors, and myrcene is one of the most important. It is a component of the essential oil of several plants including Pistacia lentiscus var. Chia [49]. It is produced mainly semi-synthetically from myrcia, from which it gets its name. It is a key intermediate in the production of several fragrances. α-Myrcene is the name for the structural isomer 2-methyl-6-methylene-1,7-octadiene, which is not found in nature and is little used [50].

Figurer 11: Myrcene Structure.

Linalol: is a naturally occurring terpene alcohol chemical found in manyflowers and spice plants with many commercial applications, the majority of which are based on its pleasant scent (floral, with a touch of spiciness). It is also

26 known as β-linalol, linalyl alcohol, linaloyl oxide, p-linalol, allo-ocimenol, and 3,7- dimethyl-1,6-octadien-3-ol [51].

Figure 12: Linalol Structure.

Limonene: is a colorless liquid hydrocarbon classified as a cyclic terpene. The more common d-isomer possesses a strong orange smell. It is used in chemical synthesis as a precursor to carvone [52].

Figure 13: Limonene structure.

2.2 Cell Cultures

For the experiments the cell line RAW264.7 was used. RAW264.7 are adherent macrophage cells transformed by the Abelson murine leukemia virus. RAW264.7 cells were grown in Dulbecco’s Modified Eagle Medium containing 10% fetal bovine serum, 100 units/ml penicillin, 100 μg/ml streptomycin and 2 mM L-glutamine. They were cultured as an adherent monolayer culture under a humidified 5% CO2 atmosphere and 37o C temperature. In order the cells to be freezed, they were trypsinised, centrifuged and re-dissolved in 1ml freezing medium (Bambanker). When the cells were grown to 80%-90% confluency they were detached by being splashed with the medium through the pipette and/or trypsinization and subcultured. Before seeding, the cells were counted using the Neubauer hemocytometer. Only the cells that were placed in the eight grid squares were counted. Cells touching the lower and right limits were not taken into account. After having gathered the counting results from the eight squares the number was multiplied with 104. The final number was the amount of the cells/ml. Cells were seeded on multiwell cell culture plates for various experiments as follows: 6x104 cells/ml in 96-well plate, 1x105 cells/ml in 12-well plate and 2x105 cells/ml in 6-well plate.

Figure 14: A big square on a Neubauer hemocytarometer.

2.3 SRB Assay

The Sulforhodamine B (SRB) assay is used for the cell density determination, based on the measurement of cellular protein content. The assay depends on the ability of Sulforhodamine B to bind to specific protein residues on the cellular surface. The cells have been immobilized by the trichloroacetic acid (TCA). SRB is a bright pink aminoxanthene dye with two sulfonic groups that binds to amino-acid residues under mild acidic conditions and dissociates under basic conditions. As the binding of SRB is stoichiometric, the amount of dye extracted from stained cells is directly proportional to the cell mass.

Experimental Procedure:

Raw cells were seeded in a density of 6000 cells/well in 100ul and they were left to adhere overnight. Without removing the cell culture supernatant, 50μl cold 10% TCA were gently added to each well. After incubation at 4oC for 1h, the plates were washed four times with slow running tap water. The excess water was removed using paper towels and a blow dryer was used to completely dry the plates. 100μl of 0.057% w/v SRB solution were added to each well using a multichannel pipettor. After incubation at room temperature for 30 min the plates were rinsed four times with 1% v/v acetic acid in order to be removed the unbound dye. A blow dryer was used again to dry the plates. Finally, 200μl of a 10mM Tris base solution (pH 10.5) were added to each well and then the plate was shaked for 5 min on a gyratory shaker to solubilize the protein-bound dye. The OD was measured at 510 nm in a microplate reader.

29 Figure 15: Plate containing SRB solution. 2.4 Griess Test

Nitric Oxide (NO) is an important mediator-molecule that participates in many biological systems such as immunological, neuronal and cardiovascular tissues. Due to its involvement in these diverse systems there is a big interest in measuring the amount of NO in the biological fluids. It is involved in innate immunity as a toxic agent towards infectious organisms but it can also induce or regulate death and function of host immune cells, thereby regulating specific immunity. One way to - measure the amounts of NO is to estimate the levels of nitrite (NO2 ) which is one of the two primary, breakdown products of NO. This assay is based on the diazotization reaction, which was discovered by Griess in 1879. Nowadays the Griess Reagent System relies on a chemical reaction in which sulfanilamide and N-1- napthylethylediamine participate under acidic conditions. The system measures the - levels of NO2 in a variety of samples such as plasma, serum, urine and tissue culture medium.

Griess Test is an analytical test which detects the presence of nitrite ion in solutions. Nitrite is detected and analyzed by formation of a red pink colour upon treatment of - a NO2 containing sample with the Griess reagent. When sulphanilic acid is added the nitrites form a diazonium salt. When the azo dye agent is added a pink colour develops.

Figure 16: Griess Reaction. (Tsikas D. Analysis of nitrite and nitrate in biological fluids by assays based on the Griess reaction: Appraisal of the Griess reaction in the L-arginine/nitric oxide area of research. Journal of Chromatography B, 2007). 30

Experimental Procedure:

Firstly, a Nitrite Standard reference curve was prepared for accurate quantitation of - NO 2 levels in experimental samples. 1ml of a 100μM nitrite solution was prepared by diluting the provided 0.1M Nitrite Standard (1:1,000) in the buffer used for the experimental samples. 2 columns were designated (16 wells) in the 96-well plate for the Nitrite Standard reference curve. 50μl of the appropriate buffer were dispensed into the wells in rows B–H. Then 100μl of the 100μM nitrite solution were added to the remaining 2 wells in row A. Immediately 6 serial twofold dilutions (50μl/well) were performed in duplicate down the plate to generate the Nitrite Standard reference curve (100, 50, 25, 12.5, 6.25, 3.13 and 1.56μM), discarding 50μl from the 1.56μM set of wells. The final volume in each well was 50μl. 50μl of each experimental sample were added to wells in duplicate. Using a multichannel pipettor, 50μl of the Griess Reagent were dispensed to all experimental samples and wells containing the dilution series for the Nitrite Standard reference curve. The incubation lasted 15 min at room temperature in dark. OD was measured at 520 nm using a microplate reader.

2.5 Enzyme Linked Immunosorbent Assay (ELISA)

ELISA is a type of analytic biochemistry assay that uses a solid-phase enzyme immunoassay to detect the presence of a substance in a liquid sample. Performing a sandwitch ELISA involves a plate that is coated with a capture antibody. Any non- specific binding sites on the surface are blocked. Then, the antigen-containing sample is applied to the plate and captured by antibody. After the plate wash for the removal of the unbound antigen, a specific antibody is added and binds to the antigen. Enzyme-linked secondary antibodies are applied as detection antibodies. The plate is washed to remove the unbound antibody-enzyme conjugates. Then, a chemical is added to be converted by the enzyme into a color or fluorescent or

31 electrochemical signal. Finally, the absorbance of the plate wells is measured to determine the presence and quantity of the antigen.

Figure 17: Sandwitch ELISA. ( ELISA Principle Basis and Extension, ELISA Encyclopedia, Sino Biological Inc.).

In this study ELISA was used for the detection and quantification of TNF-α in supernatants of Raw264.7. The protocol was based on the kit of Affymetrix eBioscience.

Experimental procedure:

A supplied ELISA plate with 100μl/well of capture antibody in 1X coating buffer was used. After incubation overnight at 4oC, wells were aspirated and washed 3 times with >250μl/well Wash Buffer. The excess buffer was removed using paper towels. 1 part of 5X ELISA/ELISPOT Diluent was diluted with 4 parts DI. The wells were blocked with 200μl/well of 1X ELISA/ELISPOT Diluent before incubation at room temperature for 1 hour. The samples were added at the volume of 100μl/well to the appropriate wells. After 5 washes, 100μl/well of detection antibody was used to seal the plate. Incubation at room temperature and 5 washes are the following steps. Then the plate was sealed with 100μl/well of Avidin-HRP before incubation at room temperature for 30 min. After 5 washes 100μl/well of 1X TMB solution were added to each well. The plate was incubated at room temperature for 15 minutes and then 50μl of Stop Solution were added to each well. The OD was measured at 450 nm in a microplate reader.

2.6 Prostaglandin E2 (PGE2) Assay

This assay is based on the forward sequential competitive binding technique in which PGE2 present in a sample competes with horseradish peroxidase (HRP)- labeled PGE2 for a limited number of binding sites on a mouse monoclonal antibody. PGE2 in the sample is allowed to bind to the antibody in the first incubation. During the second incubation, HRP-labeled PGE2 binds to the remaining antibody sites. Following a wash to remove unbound materials, a substrate solution is added to the wells to determine the bound enzyme activity. The color development is stopped, and the absorbance is read at 450 nm. The intensity of the color is inversely proportional to the concentration of PGE2 in the sample.

Experimental Procedure:

200 μL of a Calibrator Diluent were added to the non-specific binding (NSB) wells, 150 μL of Calibrator Diluent were added to the zero standard (B0) wells and 150 μL of each sample were added to the remaining wells. Then 50 μL of the Primary Antibody Solution were added to each well (excluding the NSB wells) and the plate was covered with a plate sealer. The following incubation lasted 1 hour at room temperature on a microplate shaker. 50 μL of PGE2 Conjugate were added to each well without washing the plate. The plate was covered with a new plate sealer and incubated for 2 hours at room temperature on the shaker. Each well was aspirated and washed, repeating the process three times for a total of four washes. The wash was carried out by filling each well with Wash Buffer (400 μL). After the last wash, any remaining Wash Buffer was removed by decanting. The plate was blotted against clean paper towels. Then 200 μL of Substrate Solution were added to each well. The following incubation lasted 30 minutes at room temperature on the benchtop in dark conditions. Finally, 100 μL of Stop Solution were added to each well. The color in the wells changed from blue to yellow. The optical density of each well was determined within 30 minutes, using a microplate reader set to 450 nm.

Figure 18: PGE2 Assay.

2.7 Comet Assay

The Single Cell GelElectrophoresis assay (SCGE, also known as comet assay) is a sensitive technique for the detection of DNA damage at the level of the individual eukaryotic cell. It is mostly used for its ability to measure DNA single-strand breaks although modifications of the method allow the detection of DNA double-strand breaks, crosslinks, base damage and apoptotic nuclei. According to this method cells embedded in agarose on a microscope slide are lysed with detergent and high salt to form nucleoids containing supercoiled loops of DNA linked to the nuclear matrix. Electrophoresis at high pH results in structures resembling comets that are observed by fluorescence microscopy. The intensity of the comet tail relative to the head reflects the number of DNA breaks (Figure 19). The likely basis for this is that loops containing a break lose their supercoiling and become free to extend towards the anode. This is followed by visual analysis with staining of DNA and calculating fluorescence to determine the extent of DNA damage. This can be performed by manual scoring or automatically by imaging software.

Figure 19: The extent of the DNA damage is proportional to the size of the comet’s tail. Cells that belong to Class 0 are undamaged, while cells that belong to the Class IV are extensively damaged. (Aviello G., Canadanovic-Brunet J.M., Capasso R., Fattoruso E., Taglialatela-Scafati O., Fasalino I. Potent antioxidant and genoprotective G, a rotenoid isolated from Boerhavia diffusa. PLoS One, 2011, 6: 19628).

Experimental Procedure:

 Reagents

1. Alkaline Lysis Buffer: 1.2M NaCl, 100mM Na2EDTA, 0.1% sodium lauryl sarcosinate, 0.26M NaOH (pH>13). Equilibrate at 4oC.

2. Alkaline Rinse and Electophoresis solution: 0.03M NaOH, 2mM Na2EDTA (pH~ 12.5)

 Agarose Preparation

Firstly, a water bath was equilibrated at 40oC. 1% low-melting agarose was prepared by mixing powdered agarose with distilled water in a glass beaker. Then the agarose was placed into the 40oC water bath after melting in a 100oC water bath for 20 min.

 Slide Precoating

To improve agarose adhesion the edges of microscope slides were scored. Then, the agarose precoated slides were prepared by dipping the slides into melted 1% agarose and wipping one side clean. The agarose air-dried to a thin film.

 Sample Preparation

The cells that had been treated with mastic oil or its major components were prepared in a suspension (40,000 cells/ml). We pipetted 0.4 ml of cells and 1.2 ml of low-melting agarose into a 5 ml plastic tube before mixing and pipetting 1.2 ml of cell suspension onto the agarose-covered surface of a pre-coated slide.

 Lysis and Electrophoresis

Lysis and electrophoresis were performed in alkanine conditions. After agarose gelled, slides were submerged in a covered dish containing lysis solution. The incubation lasted 1 hour in the dark at 4oC and then the slides were removed and placed in rinse solution for 20 min. The wash was repeated twice. After these two washes, the slides were submerged in fresh rinse solution in an electrophoresis chamber. The chamber was filled with a consistent volume of buffer that was about

1-2 mm above the top of the agarose. The electrophoresis was conducted for 25 min at a voltage of 12V.

 Slide staining

The slides were removed from electrophoresis chamber and rinsed twice in distilled water. Then, we pipetted 100μl of a 10μg/ml stock solution of propidium iodide directly onto the slide and incubated for 20 min.

 Slide Analysis

After the slides were observed under a fluorescence microscope, comet tails were counted and data were analyzed as follows. The data analysis was based on the separation of nuclei in five classes. This categorization relied on the size of the comet’s tail which was proportional to the extent of the damage. The five classes (0,1,2,3 and 4) were proposed by Coliins in order to be simplified the characterization of nuclei which were observed under the fluorescence microscope. In class 0 belong the nuclei that present no DNA-damage, while in class 4 are ranked the nuclei that possess the biggest DNA-damage and as a result they have the biggest tail. After the classification of 100 nuclei/sample the total damage was resulted from the following formula:

[(0x number of nuclei that belong to class 0)+(1x number of nuclei that belong to class 1)+(2x number of nuclei that belong to class 2)+(3x number of nuclei that belong to class 3)+(4x number of nuclei that belong to class 4)]

According to the above formula the total damage was between 0-400 arbitrary units. For the data analysis we considered 100% the damage caused from LPS (control). The % amount of the DNA-damage of the other samples was evaluated compared to the control.

2.8 Phagocytosis Assay

In order to investigate the effect of mastic oil and its major components on the macrophage cells’ phagocytosis rate, L. casei was used as an in vitro pathogen model. After treating RAW264.7 cells either with mastic essential oil or its bioactive constituents or DMSO (control), cells were co-cultured with stained L. casei (2X107 CFU/ml) for 4h. The cells were washed and fixed in ice-cold methanol. The observation was carried out under a fluorescence microscope. For the data analysis, RAW cells that had phagocytosed 0, 1, 2 or 3 bacteria were counted in order to determine whether mastic oil affects phagocytosis rate on RAW264.7 cells.

The following protocol was carried out:

 Reagents

Hoechst 33342 (10mg/ml)

CFSE (Carboxyfluorescein succinimidyl ester) (20μΜ)

CellBrite™ Cytoplasmic Membrane Stain

 Membrane Staining In order to be sure that the bacteria had been engulfed by the macrophage cells, the membranes of the macrophages were stained with CellBrite™ Cytoplasmic Membrane Stain for 20 min.

 Cell Cultures

Before seeding RAW cells in a 24-well plate, cover slips were putted in each well. The coverslips were firstly immerged in PBS, secondly in ethanol and thirdly again in PBS. Cells were left to adhere and grow on the coverslips overnight (37oC,

5%CO2).

 Bacterial Culture

Lactobacillus casei ATCC 393 (DSMZ, Germany) was grown in MRS Broth at 37°C without agitation. Bacteria were harvested in late-log/early stationary phase of growth (109 CFU/mL) by centrifugation at 1700 g for 15 minutes at 4°C.

 Nuclei Staining

In each well of a 24-plate there were adherent cells in a final volume of 1ml. The cell culture supernatant was discarded and Hoechst solution was gently added to each o well for 40min (37 C, 5% CO2).

 L. casei Staining

The L. casei culture was vortexed and centrifuged for 15min (4oC, 3000rpm). The pellet was washed using 3ml PBS before and centrifuged again. It was resuspended in 1ml CFSE solution and incubated for 15min in a water bath at 37oC. Then the cell culture was centrifuged and washed twice. The second wash was carried out in 1ml DMEM. The incubation lasted 30min at 37oC. The cells were centrifuged again and washed in 1ml DMEM.

 Treatment

RAW264.7 cells that grown in the 24-well plate were washed three times using 1ml PBS. The PBS was discarded and 1ml of the essential oil-treatment solution (mastic essential oil or its major components in non-toxic concentrations) was added in each well for 1h. Raw cells were co-cultured with stained L. casei (2x107 CFU/ml) for 4h in either complete DMEM (control) or essential oil–treatment o solution medium (37 C, 5% CO2).

 Fixation

The culture medium was discarded from the plate and each well was washed three times for 1 min. using 1ml PBS. The cells were fixed in ice-cold methanol for 5 min.

Methanol was discarded and cells were washed in PBS (3 x 5min). 5μl of Mowiol mounting medium were used per sample and coverslips were placed on a microscope slide. The slides could be stored in the dark at 4oC and observed under a fluorescence microscope as described below.

 Data Analysis

The pictures that were taken by the fluorescence microscope were analyzed using the programme ImageJ. After the adjustment of brightness and color the pictures were merged resulting in a final picture depicting the number of L. casei that each cell had engulfed through phagocytosis.

2.9 Fluorescence Microscopy

Τhe fluorescence microscope ZEISS, Scope A1(Figure 20) was used both in Comet and Phagocytosis Assay. The nuclei in Comet Assay were observed using the Cy5 channel, while the nuclei in Phagocytosis Assay were observed using the DAPI channel. Also, the L. casei cells became distinct through the FITC channel. In both assays the observation was carried out using the 20X lens.

Figure 20: Microscope ZEISS, Scope A1.

2.10 Optical Microscopy

The optical microscope ZEISS, Primovert (Figure 21) was used in order to observe the morphological changes in RAW264.7 cells. Observation was carried out using the 20X lens and the pictures were taken with an Axiocam ERc 5s camera.

Figure 21: Microscope ZEISS Primovert.

2.11 Statistical Analysis

Sigmaplot 11.0 was used for the statistical analysis of the results. Every experiment was conducted independently at least three times and multiple replicates were used. Results are presented as mean +/- standard deviation between the experimental repeats. Statistical significance of the results was evaluated by applying t-test or one- way ANOVA where appropriate (p<0.05). All the diagrams were created using Sigmaplot.

3. Results

3.1 Determination of the maximum non toxic concentrations of mastic oil and its major components.

Τhe maximum concentrations of mastic oil and its major components that are non toxic to RAW264.7 after a 24h treatment ,were determined with the SRB Assay. After treatment, the cells were fixed with TCA, stained with SRB dye which was solubilized with Tris-Base and the OD was measured at 510 nm. As the binding of SRB on protein is stoichiometric, the amount of dye extracted from stained cells is directly proportional to the cell mass. The concentrations are presented in Table III.

Table III: The maximum non toxic concentrations of mastic oil and its major components .

Oil/ Components Maximum non toxic concentration (mg/ml)

Mastic 0.0089

Myrcene 0.057

B-pinene 0.0623

limonene 0.0609

linalol 0.089

a-pinene 0.0087

3.2 Mastic oil and b-pinene reduce significantly the production of NO, PGE2 and TNF- α in supernatants of LPS-induced RAW cells.

As inflammatory mediators NO, PGE2 and TNF-α are produced by activated RAW macrophage cells. In order to study if mastic oil οr its bioactive constituents possess potential protective activity against inflammation we observed how the amounts of the above mediators are affected after the treatment of activated macrophages with the mastic oil or its constituents.

We first confirmed that the optimal concentration of LPS for the stimulation of NO production by RAW264.7 cells was 0.1μg/ml. LPS was added 30 min after the addition of mastic oil or its constituents. Τhe greatest reduction of the NO concentration, which was statistical significant, was observed after treatment with b- pinene (20.3943 uM, P= 0.027 vs Control) followed by α-pinene (23.9424 uM) and myrcene (25.8245 uM) compared to control (50 μM) (Figure 22).

Furthermore we found that untreated RAW264.7 cells produced a background level of PGE2 and the addition of 0.1μg/ml LPS caused an increase of PGE2 production. B- pinene (920 pg/ml), linalol (1300 pg/ml) and limonene (1500 pg/ml, P=0,001 vs

Control) seemed to reduce the production of Prostaglandin E2 compared to control

(2500 pg/ml) (Figure 23).

Figure 22: The greatest reduction of the NO concentration in the supernatants of LPS-induced RAW264,7 cells was 43 caused by b-pinene followed by mastic oil*P<0.05 vs Control.

Figure 23: Myrcene and Limonene inhibited the production of PGE2 in LPS-induced RAW cells. *P<0.05 vs Control, ***P<0.001 vs Control.

Similar to the production of PGE2, untreated RAW264.7 cells produced a background level of TNF-α and the addition of 0.1μg/ml LPS caused an increase of TNF-α production. Mastic essential oil and linalol seem to reduce the production of TNF-α. Mastic oil reduced the concentration of TNF-α at 98 pg/ml, and linalol at 278 pg/ml (P=0.03 vs Control) from 520 pg/ml (control) (Figure 24).

Figure 24: Mastic oil and linalool reduce the amount of TNF-α in the supernatants of the LPS-induced RAW264.7 cells. *P<0.05 vs Control, **P<0.01 vs Control.

3.3 Mastic oil, limonene and linalol protect against LPS-induced DNA in RAW264.7 cells.

We observed that LPS in non toxic concentrations could cause DNA damage to the cells. Comet Assay was used to study whether a pre-treatment with mastic oil or its major constituents affects the extent of DNA damage LPS causes on RAW264.7 cells. We concluded that mastic oil and some of its bioactive main constituents could inhibit LPS-induced DNA damage in RAW264.7 cells. Mastic reduces % DNA-damage at 57.8% and limonene seems to restrict the DNA damage caused by LPS at 64.1%. Also, linalol decreased the DNA damage in a statistically significant manner (P=0.003) from 100% (control) to 58.13%. α- and b-pinene do not provide any protection (Figure 25).

Figure 25: Mastic, linalol and limonene reduce the DNA damage caused by LPS in RAW 264.7 cells. **P<0.01 vs Control.

3.4 Inhibition of Morphological Changes in LPS-Stimulated RAW 264.7 Macrophages by mastic oil.

The treatment of RAW264.7 macrophage cells with LPS had as a result their activation, which is accompanied by morphological changes that can be observed using the optical microscope. Activated cells form podia and they increase in size. (Figure 26).

LPS

Figure 26: Morphological changes between the untreated and the LPS-activated RAW264.7 cells.

We observed that these morphological changes were partly inhibited in the cells that were pre-treated with mastic oil or some of its major constituents. Specifically, the rate of % differentiated/% undifferentiated cells reduced by b-pinene to 0.23%compared to control (rate of % differentiated/% undifferentiated = 1) in a statistically significant manner (P= 0.0082) (Figure 27).

Figure 27: Morphology of untreated (left), LPS-induced (middle), treated with LPS and b-pinene (right) RAW 264.7 cells . 47

Also linalol reduced the rate of % differentiated/% undifferentiated cells of RAW264.7 cells that presented LPS-induced morphological changes to 0.36 and mastic oil to 0.38 (P=0.042 vs Control).

Figure 28: B-pinene, linalol and mastic reduce the number of differentiated/undifferentiated macrophage cells.*P<0.05 vs Control, **P<0.01 vs Control.

igure 25: 3.5 Limonene, linalol and mastic oil reduce the rate of phagocytosis of L. casei by RAW264.7 cells. In order to study the phacytic rate of L. casei bacteria by RAW cells the membranes and nucleui of RAW264.7 cells and L. casei bacteria were fluorescently stained before the co-culture(Figure 29). In figure 29 the nucleus is dyed blue, the cell membrane red and the bacteria green.

Figure 29: A RAW cell phagocytosing L.casei bacteria.

Table IV shows the % number of RAW 264.7 cells that have phagocytosed 1, 2, 3 or 0 L. casei. Limοnene, linalol and mastic seem to reduce phagocytosis rate, as the number of RAW264.7 cells that are not associated with bacteria (% cells without L. casei ) is increased compared to control. The same results are represented below in another way.

Table IV: % RAW cells that have phagocytosed 1, 2, 3 or 0 of L.casei bacteria after treatment with mastic oil or its constituents-phagocytosis compared to the control.

% 1 L.casei/cell % 2 L.casei/cell % 3 L.casei/cell % cells without L.casei Control 45.09 16.58 1.33 36.5 (RAW +L.casei) Mastic 23.66 5.84 0 74 Myrcene 14.71 2.88 26.5 46.7 Linalol 12.73 4.76 0.5 82.43 α-pinene 22.8 0 21.7 55.5 Limonene 20.75 6.27 1.66 82.43 b- pinene 21.36 6.51 1.40 67.17

Linalol seems to reduce the phagocytosis rate to 17.995% in a statistically significant manner (P=0.034) (Fig 30). The reduction that caused b-pinene (27.64%) is also statistically significant (P=0.028). Furthermore limonene (28.53%) and mastic oil (27.35%) seemed to inhibit phagocytosis of L. casei bacteria by RAW264.7 cells, as cells treated with these compounds show a lower phagocytic rate.

Figure 30: Mastic oil and its constituents inhibit phagocytosis of bacteria by RAW 264.7 cells. *P<0.05 vs Control.

4.Discussion

Inflammation is an endogenous mechanism that participates in host defenses against agents and injury but it also contributes to the pathophysiology of many chronic diseases. Interactions of cells in the innate immune system, adaptive immune system and inflammatory mediators orchestrate aspects of the acute and chronic inflammation. A coordinated series of common effector mechanisms of inflammation contribute to tissue injury, oxidative stress, angiogenesis and fibrosis in diverse target tissues [53].

Pistacia lentiscus var Chia is an evergreen bush of the Anacardiacae family that extensively thrives in Chios island. Chios mastic gum is obtained after hurting the trunk and the branches of the bush [19]. Knowledge of the benefits of mastic gum consumption goes back to ancient times, when it was used for the treatment of various gastrointestinal malfunctions such as gastralgia, peptic ulcer and dyspepsia [20]. In modern times mastic gum has been used as a supplement in liquors, drinks, foods, chewing gum, toothpaste, lotions and other cosmetics [21]. Also, mastic oil because of its multiple effects on many pathways of inflammation could be used for the development of nutritional and pharmaceutical supplements that possess anti- inflammatory activity.

A-pinene was found to exhibit anti-inflammatory activity through the suppression of the NF-κΒ pathway in mouse peritoneal macrophages [45] while limonene was shown to suppress the LPS-induced production of NO, PGE2 and pro-inflammatory cytokines such as IL-1β [43]. Also linalol inhibited NO production by LPS stimulated macrophage cell line J774.A1 [54]. For this reason in this study we tried to investigate the potential anti-inflammatory activity of mastic oil and its major components on an in vitro inflammation model based on LPS-treated RAW264.7 murine macrophages.

Table V summarizes the effects of mastic oil and its bioactive constituents on the levels of TNF-α, PGE2 and NO in supernatants of LPS induced macrophage cells as described in the Results section. TNF-α seems to be reduced significantly by mastic oil. Linalol and limonene affect moderately the reduction of TNF-α. These results are

in agreement with the study by Yoon et. al. [43] where it was shown that limonene

suppresses LPS-induced NO, PGE2 and TNF-α in a dose-dependent manner in LPS induced-RAW264.7 macrophage cells. On the other hand, we observed that mastic

oil’s constituent that reduces PGE2 in a significant rate is b-pinene. Mastic, myrcene,

limonene and linalol play a secondary role in the reduction of PGE2. The third inflammatory mediator, NO, is being reduced importantly by b-pinene too. Myrcene and α-pinene decrease impalpably NO production, while mastic linalol and limonene do not seem to affect it.

Table V: Inhibition of TNF-α, PGE2 and NO production by LPS-stimulated RAW264.7 cells treated with mastic oil or its major components. +++: significant reduction, ++: medium reduction, +: minimum reduction, -: no effect

TNF-α PGE2 NO

Mastic +++ + _

Myrcene _ + +

Linalol ++ ++ _

α-pinene - + +

Limonene ++ ++ -

b- pinene + +++ +++

Moreover, mastic oil, linalol and limonene showed to protect RAW264.7 cells against DNA-damage caused by LPS. Treatment with b-pinene, α-pinene or myrcene did not inhibit LPS induced DNA damage. Linalol was also shown to inhibit the DNA-damage in the study by Coelho et. al. Cells of the peripheral blood isolated from mice that were treated with linalol, show no increase in DNA damage caused by hydrogen peroxide [55].

Moreover, we observed that linalol and b-pinene inhibit phagocytocis of L. casei bacteria by RAW264.7 cells compared to control untreated cells.The study by da Silveira e Sa et. al. is in agreement with the previously observed inhibition of phagocytic and microbicidal activity of murine peritoneal macrophages by many monoterpenes [56]. Finally, b-pinene and mastic oil significantly reduced the amount of RAW264.7 cells that underwent LPS-induced morphological changes. This reduction reflects also reduction to the number of the activated macrophage cells that are recruited during inflammation.

Considering all the above results, probably the modulation of the inflammatory response in murine macrophages relies on the synergistic activity of all its constituents. B-pinene seems to play a central role as it participates in the inhibition of phagocytosis of L. casei by RAW264.7 cells, the reduction of the amount of RAW264.7 cells that undergo LPS-induced morphological changes and the inhibition of production of NO and PGE2.

Further experiments should be conducted, using an in vitro model based on human cells , such as the human THP1 cell line. Moreover, in vivo experiments in murine inflammation models such as the carrageenan-induced paw edema could help to further evaluate the anti-inflammatory and immunomodulatory activities of mastic oil.

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