Abstract

Curcuma longa and Cinnamon are used in folkloric medicine and thought to have different pharmacological activities including anti-inflammatory effects. The objective of this work is to evaluate the in vitro inhibitory effect of Curcuma longa and Cinnamon ethanolic extracts on Lipopolysacaride (LPS)-induced Interlukin-6 (IL-6) and Tumor Necrosis Factor-α (TNF-α) by polymorphonuclear Cells (PMNCs). Polymorphonuclear cells were isolated from the whole blood using Histopaque (Ficol-1077) method and then cultured in an enriched Roswell Park Memorial Institute (RBMI) medium. The concentrations of TNF-α and IL-6 in the supernatant were measured after 24 h and compared using paired-samples t test. The Curcuma longa and Cinnamon extracts have shown significant reduction in the levels of both Il-6 and TNF-α. HPLC analysis of Curcuma longa extract revealed that it contains curcumin, demethoxycurcumin, and bisdemethoxycurcumin while the major compound in the extract of cinnamon was found to be cinnamic acid. Reduction in the levels of IL-6 and TNF-α upon effect of the plants” extract is an indication of their anti-inflammatory effects. The observed anti-inflammatory effect may be due to the presence of curcuminoids and cinnamic acid from Curcuma longa and Cinnamon, respectively.

1. Introduction

Cinnamon and curcumin are among the herbal plants that have been used in many different cultures to treat patients who suffered from different diseases. They have pharmacological activities such as anticancer, anti-inflammatory, antimicrobial and antioxidant activities ^{1, 2, 3}.

Curcumin is a pigment with yellow color commonly used as a food coloring agent and found in turmeric roots, mostly planted in India and widely used to cure many diseases such as hepatic disorders, rheumatism, skin diseases, dyspepsia, blood sugar, colic inflammation, and amenorrhoea ⁴. Due to its highly effective properties, many companies are currently providing curcumin in the form of capsules, soft drinks, gels, nasal sprays, and tablets ⁵. Cinnamon (Cinnamomum zeylanicum) contains a high proportion of eugenol which has antispasmodic, antiparasitic, antibacterial, and antidiarrheal effects. Thus, such herb has been used to treat many disorders. Also, it has a wound healing activity ^{6, 7}.

Lipopolysaccharide (LPS) is an endotoxin and a part of the cell wall of the gram negative bacteria. It is important to the integrity of the bacterial structure and helps in protection of the bacteria from attacks by certain chemicals. LPS enhance the immune response of the immune system by binding to Toll Like Receptor 4 (TLR 4) in many immune cells, as Dendritic cells, B cells, Monocytes and Macrophages ^{8, 9, 10}.

Interlukin-6 and Tumor Necrosis Factor – α are proinflammatory mediators that are released from many immune cells as Monocytes, B cells, and Macrophages upon exposure to an inflammatory signal such as the exposure to LPS ^{11, 12, 13}. Anti-inflammatory agents decrease the production of these pro-inflammatory cytokines (IL-6 and TNF-α).

Many investigations have been conducted to clarify the anti-inflammatory effect of curcumin as well as cinnamon ^{5, 14, 15, 16, 17, 18, 19}. In this research, the anti-inflammatory effect of Curcuma longa and Cinnamon was evaluated using LPS-induced mononuclear cells isolated from the buffy coat of whole blood. IL-6 and TNF-α were measured before and after the addition of the LPS and the extracts ^{20, 21}. Levels of the cytokines were measured using Enzyme Linked Immune Sorbent Assay (ELISA) method. Analysis of the active compounds of Curcuma longa and Cinnamon extracts by HPLC-UV was also conducted in this study.

2. Materials and Methods

Grinded cinnamon and curcumin were purchased from Alsirisi market, Jenin, Palestine and classified by Mostafa Amarni (botanical specialist at the Ministry of Agriculture, Jenin, Palestine). Cinnamon identification number is 09061100 and curcumin identification number is 969516.

Fifty gm of powder of cinnamon and 50 gm powder curcumin were mixed separately in 500 ml of 96% ethanol and kept shaking for one week at room temperature. The solution was filtered using a Whatman filter paper and the filtered product was evaporated using rotary evaporator (BUCHI brand) under vacuum at 50°C. The extract was collected and kept at -20°C until further use.

Polymorphonuclear cells were collected from a 5 ml whole blood by a method as described by Qabaha et al ²². The cells were cultured in Roswell Park Memorial Institute medium (RPMI) enriched with 100 Uml^-1 penicillin, 100-μg ml^-1 streptomycin, and with 10% heat-inactivated Fetal Bovine Serum (FBS). The mixtures were divided into a 12 well tray in 5% CO₂ incubator at 37°C for 24 hour. Each well contains one ml of the mixture which contains one million cells. Wells were exposed to different concentrations of the plant extracts in the presence and absence of Lipopoly Sacharide (LPS) (1 μg/ well).

Cytotoxicity of the curcumin and cinnamon extract was evaluated using Trypan Blue Exclusion Test as described by Avelar-Freitas et al. 2014 ²³. The dead cells will be stained with trypan blue while the viable cells will be colorless.

Enzyme Linked Immunoassay was used to quantify TNF-alpha and IL-6 according to manufacturer’s instructions.

The analytical HPLC is Waters Alliance (e2695 separations module), quipped with 2998 Photo diode Array (PDA). Data acquisition and control were carried out using Empower 3 chromatography data software (Waters, Germany).

The HPLC analytical experiments of the crude water, 80% ethanol and 100% ethanol extracts were run on ODS column of Waters (XBridge, 4.6 ID x 150 mm, 5 μm) with guard column of Xbridge ODS, 20 mm x 4.6mm ID, 5 μm. The mobile phase is a mixture of 0.5% acetic acid solution (A) and acetonitrile (B) ran in a linear gradient mode. The start was a 100% (A) that descended to 70% (A) in 40 minutes. Then to 40% (A) in 20 minutes and finally to 10% (A) in 2 minutes and stayed there for 6 minutes and then back to the initial conditions in 2 minutes. The HPLC system was equilibrated for 5 minutes with the initial acidic water mobile phase (100 % A) before injecting next sample. All the samples were filtered with a 0.45 μm PTFE filter. The PDA wavelengths range was from 210-500. The flow rate was 1 ml/min. Injection volume was 20 μl and the column temperature was set at 25°C. The HPLC system was then equilibrated for 5 minutes with the initial mobile phase composition prior injecting the next sample. All the samples were filtered via 0.45 μm micro porous disposable filter.

3. Results and Discussion

Curcuma longa (turmeric) has a significant influence as an anti-inflammatory agent. Turmeric constituents include three major curcuminoids, namely, curcumin which is responsible for its yellow color, demethoxycurcumin, and bisdemethoxycurcumin, see Figure 1.

Upon injection of the ethanolic extract to analytical HPLC chromatograph, it has been noticed that the curcumin extract comprises mainly the three major curcuminoids (Figure 2), while there were only negligible amount of phenolics in the range between 3-45 minutes when compared to standard phenolic mixture which ran under the same chromatographic conditions at the same monitoring wavelength (Figure 3).

Therefore, the anti-inflammatory influence is probably due to these three major curcuminoids compounds.

The Cinnamon ethanolic extract revealed one major peak eluted at 36.7 minutes along with some other minor ones at 290 nm. Standard cinnamic acid was injected and the retention time and UV-Vis were matched exactly with cinnamic acid from the extract indicating its identity (Figure 4). This major peak with the other minor existing peaks did not match any of the phenolic and flavanoidic standard mixture retention or UV-Vis as shown in Figure 5. It is reported that cinnamic acid derivatives and esters have a tangible anti-inflammatory effect ^{24, 25}. This also strengthens the role cinnamic acid plays as an anti-inflammatory agent.

Curcumin and Cinnamon extracts at concentration 300 μg/ ml and LPS at concentration of 1 μg/ ml have no negative effect on the viability of the Poly Morpho Nuclear cells as shown by Table 1.

Upon stimulation of PMNCs with 1 μg/ ml LPS, the production of IL-6 and TNF-α has increased significantly after 24 hour. However, after treatment with the plant extracts, the production of both cytokines has decreased as the concentration of the plant extract increased in a dose-dependent manner (Table 2). Furthermore, the differences in the inhibition of the cytokine production upon the effect of each dose were significant (different small letters within column indicate significant difference.

Poly morpho nuclear cells play an important role in host defense as well as in inflammation. LPS- stimulated PMNCs secrete many pro-inflammatory mediators such as TNf-α and IL-6. Many disease causative agents secrete LPS that in turn initiate inflammation in the host. In our study, we mimicked the disease by adding LPS to the PMNCs to release pro-inflamatory mediators.

In order to evaluate the antiinflamatory effect of the extract of cinnamon and curcumin, different concentrations of both extracts were added on the LPS-stimulated PMNCs and both TNf-α and IL-6 levels were measured after 24hours of incubation at 37C in 5% CO2 incubator.

It was found that cinnamon extract has stronger effect in the inhibition of the release of TNf-α compared to curcumin, while the reverse was observed for IL-6 where Curcumin showed stronger effect in the inhibition of IL-6 release compared to cinnamon, Table 2.

The anti-inflammatory effects of both Curcuma longa and Cinnamon are consistent with the findings of Jurenka ¹⁴, Menon and Sudheer ¹⁵, Jacob et al. ²⁶, Mashhadi et al ¹⁶, Hong et al. ¹⁷, Joshi et al. ¹⁸, and Rao et al. ¹⁹.

4. Conclusion

Curcuma longa and Cinnamon extracts showed strong reduction in the production of IL-6 and TNF-α from the LPS-induced PMNCs, indicating strong anti-inflammatory effects, which make Curcuma longa and Cinnamon a source of anti-inflammatory candidates to be used in the pharmacological industry. Both extracts at concentration of 300 μg/ml were not toxic to the PMNCs which show that these extracts are able to inhibit IL-6 and TNF-α production while maintaining cell viability. The anti-inflammatory effects of Curcuma longa are attributed to its main three compounds (curcumin, demethoxycurcumin, and bisdemethoxycurcumin), while this activity for Cinnamon is attributed to cinnamic acid present in cinnamon.

Statement of Competing Interests

The authors have no competing interests.

List of Abbreviations and Nomenclature

LPS: Lipopolysacaride

IL-6: Interlukin-6

TNF-α: Tumor Necrosis Factor-α

PMNCs: Polymorphonuclear Cells

HPLC: High Performance Liquid Chromatography

ELISA: Enzyme Linked Immune Sorbent Assay

RPMI: Roswell Park Memorial Institute medium

FBS: Fetal Bovine Serum.

References