Herbal therapies have been used successfully in treating inflammatory diseases like arthritis. Traditional Chinese and Indian medicine provide several examples of herbal remedies for arthritis treatment and bioprospecting based on traditional herbal drugs known to be effective in treatment of arthritis offers the novel approach. Cinnamomum zeylanicum commonly used as spice and well-known medicinal plant is evaluated for its anti-inflammatory activity. In vitro & in vivo experiments were performed targeting TNF-a using flow cytometry. Ethanol extract of Cinnamon showed suppression of intracellular release of TNF-a from murine neutrophils as well as from pleural fluid. The extract was found to inhibit TNF-a gene expression in LPS-stimulated human PBMCs at 20µg/ml concentration. A potent anti-inflammatory activity of Cinnamon extract is suggestive of its anti-arthritic activity, which could be confirmed in various models of arthritis.
Index entries: Cinnamomum zeylanicum, TNF-a, Flow Cytometry, Anti-inflammatory, gene expression,
Cinnamomum zeylanicum (Lauraceae) originates from the island of Sri-Lanka (formerly called Ceylon) southeast of India. Cinnamon has been used for its anti-diabetic, anti-nociceptive, astringent and diuretic activities. Cinnamomum zeylanicum fruit extract is a good source of antioxidant and antimutagenic phenolics (1-2). Cinnamomum cassia has anti-inflammatory activity and reported to inhibit inflammatory mediators such as inducible nitric oxide synthase (iNOS) (3), however, not much is known about C. zeylanicum is unexplored. Present investigation was carried out to evaluate the anti-inflammatory potential of hydroalcoholic extract of Cinnamomum zeylanicum (OA4-50) and its effect on TNF-a secretion and gene expression.
Inflammation is a process involving multiple factors acting in a complex network. The ingress of leukocytes into the site of inflammation is crucial for the pathogenesis of inflammatory conditions. Neutrophils and macrophages are known to recruit & play pivotal roles in acute & chronic inflammation, respectively (4). Recruited cells are activated to release many inflammatory responses, causing a change from the acute phase of inflammation. Therefore; inhibition of the cellular reactions is one of the targets that are generally used as an in vitro model for anti-inflammatory testing.
Tumor necrosis factor- a (TNF-a) is a pro-inflammatory cytokine, mainly produced by activated monocytes and macrophages. Excessive production of TNF- a is believed to underlie the progression of many serious inflammatory diseases, such as rheumatoid arthritis (RA), Crohn's disease and psoriasis. Therefore, anti- TNF-a therapy would be a possible tool for treatment of acute & chronic inflammatory diseases (5-6)
Materials and Methods
Histopaque-1077 density gradient solution was obtained from Sigma-Aldrich, sterile phosphate buffer saline, Trypan blue (0.4%), RPMI-1640 medium, HEPES, glutamine, fetal bovine serum, penicillin, streptomycin, TNF-a enzyme-linked immunosorbent assay kit was purchased from R & D system, cDNA synthesis kit from In Vitrogen, SYBR Green RT-PCR reaction kit from ABI, dimethylsulphoxide, ethylene diamine tetra acetic acid, rolipram (sigma), lipopolysaccharide (sigma), ? cargeenan typeIV (sigma), phycoerythrin labeled mouse TNF-a monoclonal antibody from BD, Bioscience, FACS lysing solution (BD, Bioscience), permeabilizing solution (BD, Bioscience), Golgi plug (BD, Bioscience), tolene, ethylacetate, ethanol.
Plant material & Extract preparation
Cinnamomum zeylanicum bark was collected from Green pharmacy and authenticated by routine pharmacognostic procedure. Plant material was dried under shadow and extraction is carried out by cold maceration. Dried coarse powder was macerated overnight with 50% ethanol in incubator shaker at 20C at 60 rpm. The extract was then filtered & centrifuged at 800g for 10 min. Solution was allowed to dry by lyophilisation. The percent yield was calculated on the basis of the dried plant material weight for testing. The extract was dissolved in vehicle & diluted to the desired concentration.
HPTLC characterization of the cinnamon bark extract was done. Presence of volatile oil in the extract was confirmed during preliminary phytochemical screening of the extract. The hydro-alcoholic extract was characterized by HPTLC (F254-Merk-silica gel plates) using reported TLC method for volatile oil (7). Chromatographic separation was carried out using solvent tolene: ethyl acetate (9:1.5) & plates were scanned at 225nm & 365nm (Camag Multi wavelength scanner III, version IV). The spots were developed with vanillin-sulphuric acid and scanner at visible range (wavelength 300-800). Stock solution was prepared by dissolving the extract in 50% ethanol (26.27ng/µl). Standard was prepared by dissolving the extract in 50µl, then dissolve in 10ml of ethanol. Injection volume was 5µl (500ng/spot). From calibration curve it was observed that concentration of major component (cinnamaldehyde) in extract was 2.3%
Biological activity testing
Swiss albino mice and Wistar rat were obtained from the Indian institute of Integrated medicine, Jammu. They were kept in standard environmental conditions and maintained on a standard rodent diet with water given ad libitum.
Isolation of human peripheral blood mononuclear cells (hPBMCs)
After obtaining written informed consent, venous blood sample was obtained from healthy adult donors. hPBMCs were isolated from heparinized blood samples using histopaque density gradient, centrifuged & were suspended in complete RPMI-1640 medium supplemented with 10mM HEPES, 2mM l-glutamine ,10% FBS, 100U/ml of penicillin, 100µg/ml of streptomycin. Cells were maintained at 37°C in a humidified incubator under an atmosphere supplemented with 5% CO2.
The stock solution of extracts & pure compound were prepared in DMSO & then diluted to the desired concentration in which the final maximum concentration of DMSO in the media was not more than 0.1% DMSO. Cells treated with lipopolysaccharide alone were pretreated with 1g/ml LPS in media containing 0.1% DMSO.
Quantitation of cytokines
Supernatants were collected for cytokine analysis. Cytokine levels were quantitated using ELISA kit from R & D systems, according to the manufacturer's instructions. Supernatant (100µl) was added to antibody-coated polystyrene wells and incubated for 2h. After washing, the plates were incubated with biotin-labeled anti-cytokine antibody for 2h. The plates were washed & incubated for 20 min with a streptavidin/horseradish peroxidase conjugate. The plates were washed & incubated with trimethylbenzidine and peroxide, to detect the horse radish peroxide. The reaction was stopped by the addition of 2M sulphuric acid & the absorbance read at 540 & 450 nm on a titertek Multiskan MCC/340 microplate reader (8).
hPBMCs were washed in 6-well plates twice with 1ml of sterile-ice cold PBS and lysed directly with 1ml of monophasic lysis reagent. RNA concentration and integrity were determined using Biophotometer and formaldehyde gel (9).
The mixture (20µl) contained 1µg total RNA, 1µl oligo(dt) primer, 2µl dNTP mix, 0.1M DTT, 1µl RNase out, 1µl thermoscript RT(in vitrogen) in 5X cDNA synthesis buffer. The synthesis reactions were preceded at 50° C for 60 sec & 85° C for 20min.
The primers were designed using complete cDNA sequence of human TNF-a gene available at NCBI (accession number NM_00594). The sequence (5' to 3') of the forward primer & reverse primer for amplification of Human TNF-a gene are A G C C C A T G T T G T A G C A A A C C and T G A G G T A C A G G C C C T C T G A T respectively.
Real Time Polymerase chain reaction
Reaction mixture (50µl) contained 10µl of RNA-derived cDNA, 1µl each of forward primer & reverse primer with 2X of 25µl SYBR Green PCR master mix (ABI kit). The reactions were performed in 96-well plates in a Opticon 2 real time PCR instrument. The thermal cycle conditions were as follows 30 seconds at 94°C, 30 seconds at 50°C, 30 seconds at 72°C for denaturation, annealing & polymerization reaction for 40 cycles. Fluorescence signals measured during amplification were considered positive if the fluorescence intensity was >20-fold greater than the standard deviation of the baseline fluorescence. The CT method of relative quantification was used to determine the fold change in expression (9).
Cell viability assay
Viability of cells were determined by MTT colorimetric assay after removing the supernatant, treated & control cells were incubated with MTT (5mg/ml) for 4hrs at 37 C & solubilized in DMSO. The amount of formazan production was determined spectrophotometrically at 545nm.
Flow Cytometric study (10-11)
OA4-50 was prepared as homogenous suspension in 0.1% gum acacia. Rolipram was used as standard drug for comparison & validation of test model applied.
In Vitro experiment
Neutrophils were separated from whole blood taken from retro-orbital plexus of normal swiss albino mice; added 2.5µl of protein transport inhibitors, centrifuged & histopaque was added, incubated for 10 min & then centrifuged, added FACS lysing solution. After subsequent washing with PBS, acquisition was done on the flow cytometry.
TNF-a estimation from neutrophils
LPS was added to the isolated murine neutrophils . Drug sample at different doses was added & incubated for 3hrs at 37 0C with 5% CO2 concentration. After incubation permeabilizing solution (10X) was added. Sample was thoroughly washed with PBS and spun at 900 rpm. Anti-TNF-a monoclonal antibody was added & analyzed by flow cytometer ( BD LSR) .
In Vivo studies were carried out using Pleurisy model for assessment of effect of OA4-50 extract on inflammatory parameter TNF-a. Wistar rats having a weight range of 120-180gm were employed for the study. Drug was administered orally using metal drug feeding cannula at the concentration of 5, 10, 20mg/kg, 1h before injection of 0.5ml carageenan into the pleural cavity of rat. Then after 4 hrs pleural fluid was taken out into the heparinized tubes & was processed for the detection of TNF-a expressed in the leucocytes using flow cytometer.
TNF-a estimation from pleural fluid
Pleural fluid (50µl) was taken in heparinized tubes & Golgi stop was added. Then it was treated with FACS lysing solution for the elimination of red blood cells if any. After 3 washes with PBS, 500µl of permeabilizing solution was added & incubation was carried out for 15 min. Then samples were incubated for 30 min after addition of anti-TNF-a monoclonal antibody & were analyzed on flow cytometer (BD-LSR) using Cellquest Pro software.