1. INTRODUCTION:

Inflammatory response is the defense mechanism of tissues for internal homeostasis against internal stimuli such as the production of metabolites inside the body through various pathways including external stimuli or bacterial infections¹. The primary and secondary mediators of various intracellular inflammatory regulators including such as TNF-α, IL-1β, prostagrandin, lysosomal enzymes and free radicals are involved in the inflammatory response². In particular, the transcription factors of inflammatory response are activated by stimuli such as TNF (tumor necrosis factor)-α and LPS (lipopolysaccharide), which are cytokines secreted from macrophages. This induces the expression of inducible nitric oxide synthase(inos) and cyclooxygenase-2(cox-2) and the secretion of nitric oxide and prostaglandin E2 causes inflammation^3,4.

The excessive production of NO, a vasodilator, increases inflammatory response, causes septic shock by excessive vasodilation, inhibits wound healing, and damages nerve tissue, and thus causes different diseases in the body^5,6.Steroids and nonsteroidalantiinflammatory drugs (NSAIDs) used as therapeutic agents for acute and chronic inflammatory diseases are used in various fields, but they are difficult to use for a long time because of serious concerns about side effects^7,8. Recently, botanical aroma oil that can replace these agents has been widely used as raw material of medicines for treating diseases and cosmetics, and flavoring agents^9,10.In particular, it is used for an alternative medical treatment of respiratory diseases such as allergic rhinitis, asthma, and skin diseases^11,12. Everlasting oil is extracted from helichrysumitalicum,which belongs to the Asteraceae¹³, It has been used for thousands of years as a traditional medicine in many European. A variety of experimental studies have identified potent efficacy in reducing inflammation due to several mechanisms such as inflammatory factor inhibition, Antioxidant^14,15.

Over the past several years, several studies have been conducted to study the pharmacological uses of everlasting extracts and to find various therapeutic areas. Everlasting extracts can be used in many ways to promote health and they are most commonly used to treat wounds, infections, digestive disorders and respiratory diseases, and to aid nervous system and heart health^16,17. This paper was carried out to see the secretion of NO of everlasting oil, which is used as anti-inflammatories in various fields,and the production of TNF-α , IL-1β, which are cytokines,and to examine various applications for basic physiological activity data and functional materials to demonstrate anti-inflammatory properties of everlasting oil¹⁸.

2. MATERIALS AND METHODS:

2.1 Experimental Material:

Everlasting oil used in an experiment was 100% pure natural essential oil, which was certified by an organic certification body (ECOCERT-F-32600). It used products manufactured by NEW DIRECTIONS LABORATORY LTD. Ethanol and everlasting oil were diluted to 4:1 and added to a medium.

2.2RAW 264.7 Cell culture:

RAW264.7 mouse macrophage cell line was ordered from KCLB (Korea Cell Line Bank, Korea) and used for the experiment. DMEM medium was used for cell culture and medium containing 12% FBS and 1.5% penicillin-streptomycin was used. The macrophages were cultured in a CO²incubator (37°C, 5% CO₂) and subcultured every other day. Mouse macrophage cells were washed twice with fresh medium and used with 10ug/ml LPS.

2.3 Cytotoxicity:

Toxicity of everlasting oil to cells was measured using Desai's method¹⁹. Cytotoxicity assays are to determine the degree of toxicity by measuring the conversion of MTS into formazan by mitochondrial dehydrogenases using MTS assay method. After RAW264.7 macrpphagecells were plated at 1.0×10⁶ cells in 96-well and cultured during 20 hours, everlasting oil was treated at 5 μg/mL to 500 μg/mL and cultured in CO₂ incubator for 24 hours. After 20 μl of MTS solution was added 24 hours later and reacted in CO₂ incubator (37°C, CO₂5%) for 5hours, The absorbance at 450 nm was measuredand then cell viability, which could confirm the cytotoxicity of the control group, was expressed as a percentage.

2. 4Measurement of NO:

The NO concentration was measured in the nitrite concentration using Griess reagent system^20,21. RAW 264.7 macrpphage cells were plated in 96 wells at 1.0 x 106 density and incubated for 20 hours. The cell were pretreated with everlasting oil 5ug/mL to 500ug/mL and stimulated with LPS 10ug/mL for 24 hour. The same amount of Griess Reagent was added to the medium and cultured at room temperature. The absorbance at 540 nm was measured. Measurementconcentration of sodium nitrite was used to determine NO concentration in the culture medium.

2.5Effect of TNF-α, IL-1β:

RAW 264.7 mousemacrpphage cells were plated in 96 wells at 1.0 x 10⁶ density and incubated in a CO₂incubator for 20 hours. Then, everlasting oil was treated with 5 μg / mL to 500 μg / mL.After incubation for 24 h in a CO2 incubator. TNF-α and IL-1β, proinflammatory cytokines contained in the culture medium, were measured using an ELISA

2.6 Statistical analysis:

This study was performed using students' t-testwhich were calculated as mean ± standard error (Mean ± SE) and the significance of the group was confirmed when p <0.05.

3. RESULTS AND DISCUSSION:

3.1 Influence on cytotoxicity ofeverlasting oil to RAW264.7 cells:

To confirm the cytotoxicity of everlasting oil, which is known to be nontoxic, RAW 264.7 mouse macrophagecells were used with 5 ug/mL to 500 ug/mL of everlasting oil and then MTS assay was performed. Cell viability was measured at different concentrations. As a result, no toxicity was observed up to a concentration of 500 ug/mL. The following experiments were performed at concentrations of 5 μg/mLto 500 μg/mL, which did not affect cytotoxicity RAW 264.7 mouse macrophage cells. As a result, everlasting oil showed more than 98.6±5%, 95.9±3.02% and 94.0±2.97%, respectively, at the concentrations of 5 μg/mLto 500μg/mL, which had no cytotoxicity in figure 1.

Figure 1.Influence on cytotoxicity of RAW cells

3.2 Effect of Everlasting Oil on NO secretion:

Effect of everlasting oil on NO secretion of RAW 264.7 mouse macrophage cells was measured using LPS, which is used as an inflammation inducer in Figure 2. As a result, the concentration of NO was very low in the control group in which only RAW 264.7 cells were cultured, while the concentration of NO in the group treated with LPSwas significantly increased. Treated with everlasting oil, the secretion of NO was inhibited in a concentration dependenceand significant inhibition was observed at 500 μg/mL.

Figure 2.Influence effect on NO production in RAW cells.

3.3 Effect of Everlasting Oil on TNF-α ,IL-1β secretion:

To see the effects of everlasting oil on the secretion of TNF-α , IL-1β, RAW 264.7 mouse macrophages were treated with LPS (10 ug / mL) alone, or with LPS and everlasting oil at 5 μg/mL to 500 μg/mL.After treatment with everlasting oil, the secretion of TNF-α,IL-1β were observed.According to the investigation results, the secretion of TNF-α, IL-1β was inhibited in a concentration dependence, but there was no great difference in IL-1β. However, TNF-α secretion in the experimental group treated with 500 ug/mL was significantly inhibited inFigure 3, 4.

Figure 3.Inhibitory effect on TNF-α production in RAW cells.

Figure 4.Inhibitory effect on IL-1β production in RAW cells.

4. CONCLUSION:

MTS assays were performed to measure the toxicity of everlasting oil through LPS stimulation to RAW 264.7 macrophages. There was no cytotoxicity in the control group and the experimental group treated with everlasting oil for 24 hours.

Different inflammatory regulators in cells, as primary and secondary mediators, are involved in inflammation expressed as a defense mechanism in vivo against external infections through various pathways or internal and external stimuli by metabolites in vivo. They are also responsible for different inflammatory diseases such as allergies, atopy, arthritis, heart disease, brain cardiovascular disease and disorders, and cancer[11]. Inflammation is a body defense mechanism, manifesting symptoms and signs in various ways as the most important mechanism in the body defense mechanisms.

The efficacy was demonstrated through an experiment regarding anti-inflammatory properties using various components and preparations extracted from plants. Inflammation involves a variety of mediators, and in particular, pro-inflammatory cytokines produced from cells such as activated lymphocytes and macrophages include IL-1β,TNF-α. TNF-α plays an important rolein regulating innate immune, as a major mediator of LPS stimulation. TNF-α is produced from Macrophages and Mast Cell and associated with chronic inflammation in vivo, and it shows intracellular toxicity in tumor cells. In the experimental group treated with everlasting oil to investigate the change in the inhibition of IL-1β ,TNF-α secretion, it was observed that the production of NO, TNF-α, IL-1β was inhibited in a dose-dependent manner. According to these study results, everlasting oil has significant anti-inflammatory properties due to pro-inflammatory Cytokine TNF-α production inhibitory effects in LPS-induced inflammatory model.

To confirm the cytotoxicity of everlasting oil, which is known to be nontoxic, RAW 264.7 mouse macrophage cellswere processed 5 ug/mL to 500 ug/mL of everlasting oil and then MTS assay was performed. Cell viability was measured at different concentrations.The effect of everlasting oil on NO secretion of RAW 264.7 cells was measured using LPS, which is used as an inflammation inducer in Figure 2.By looking at the effectof everlasting oil on the secretion of proinflammatory cytokine TNF-α, IL-1β,the secretion of TNF-α, IL-1β was inhibited in a concentration dependence, butIL-1β showed no significant difference. However, TNF-α secretion in the experimental group treated with 500 ug/mL was significantly inhibited in Figure 3, 4.

In conclusion, this study can be used as a basic data to objectively demonstrate the physiological activity of immunological mechanism associated with the anti-inflammatory action of everlasting oil, but it is necessary to conduct in-depth studies on anti-inflammation.

5. ACKNOWLEDGMENT:

This study was supported by the Research fund from Nambu University, 2016

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Received on 13.12.2017 Modified on 16.02.2018

Research J. Pharm. and Tech 2018; 11(7): 3003-3006.

DOI: 10.5958/0974-360X.2018.00553.X

Pro-inflammatory Cytokine Production Inhibitory Effects of Everlasting Oil

Figure 1.Influence on cytotoxicity of RAW cells