Anti-Inflammatory Activity of Pistacia khinjuk in Different Experimental Models: Isolation and Characterization of Its Flavonoids and Galloylated Sugars

Abstract

The present study aimed at isolating and elucidating the structure of the main components of Pistacia khinjuk L. and exploring its potential anti-inflammatory effect in different experimental models. The extract was evaluated for anti-inflammatory activity by measuring paw volume in three experimental models. Then, prostaglandin E₂ (PGE₂) level, ear edema, tissue myeloperoxidase (MPO) activity, histopathology, nitric oxide (NO) level, and tumor necrosis factor-α (TNF-α) level were assessed. Seven phenolic compounds, mainly flavonoids and galloylated compounds, were isolated from the aqueous methanol extract: gallic acid (1), methyl gallate (2), quercetin-3-O-β-d-⁴C₁-galactopyranoside (hyperin) (3), myricetin-3-O-α-l-¹C₄-rhamnopyranoside (myricitrin) (4), 1,6-digalloyl-β-d-glucose (5), 1,4-digalloyl-β-d-glucopyranoside (6), and 2,3-di-O-galloyl-(α/β)-⁴C₁-glucopyranose (nilocitin) (7). The anti-inflammatory activity was evidenced by decreased carrageenan-induced rat paw edema and PGE₂ elevation. In the croton oil–induced ear edema model, MPO activity was significantly inhibited, and inflammatory histopathological changes were ameliorated. In the rat air pouch model, NO generation and TNF-α release were significantly inhibited. The isolation and nuclear magnetic resonance spectral data of compound 6 from the genus Pistacia are revealed for the first time. Also, P. khinjuk L. aqueous methanol extract possesses anti-inflammatory activity in several experimental models.

Introduction

P istacia is a genus of 10 species in the Family Anacardiaceae, native to Iran, Turkey, Afghanistan, Greece, and the whole Mediterranean region. Pistacia species have attracted the attention of researchers because of their reported biologically active constituents, such as hydrolyzable tannins,¹ condensed tannins,² flavonoids,³ phenolic acids,⁴ tetracyclic triterpenes,^5,6 anthocyanins,⁷ alkaloids,⁸ and essential oils.⁹ They have been shown to exhibit antioxidant, antimicrobial, anti-inflammatory, and cytotoxic activities.^1,3,10
–12 Pistacia khinjuk L. is one of the major species that has been cultivated in Egypt. Nothing could be found in the literature concerning the biological activity and the phytochemical content of this plant.

Inflammation is an essential protective process that preserves the integrity of the organisms against physical, chemical, and infective insults.¹³ However, the inflammatory response to several insults could erroneously lead to normal tissue damage.¹⁴ Prostaglandins (PGs), thromboxanes, and leukotrienes belong to the class of prostanoid fatty acid derivatives of arachidonic acid.¹⁵

One of the early cellular events in inflammation is the margination of leukocytes, primarily neutrophils. This response can be measured by using the neutrophil-specific enzyme myeloperoxidase (MPO), an indicator of neutrophil accumulation.¹⁶ In addition, nitric oxide (NO) plays an important role in inflammation, and NO synthase inhibitors can reverse several classic inflammatory symptoms.¹⁷ Tumor necrosis factor-α (TNF-α), a cytokine, plays an important role in inflammation because of its ability to stimulate cytokine release from neutrophils and biosynthesis of chemokines.¹⁸

Currently, both steroidal and nonsteroidal anti-inflammatory drugs (NSAIDs) are used in the treatment of inflammatory disorders. Steroids have an obvious role in the treatment of inflammatory diseases but, because of rate-limiting toxicities, are only prescribed over short periods except in very severe cases where the risks are acceptable. Prolonged use of NSAIDs is also associated with severe side effects, notably gastrointestinal haemorrhage.^19,20 The recently developed cyclooxygenase-2-selective drugs have been introduced into therapy but, however, do not seem to be free of risk.²¹ Consequently, there is a need to develop new anti-inflammatory agents with minimal side effects. In this regard, natural products have long gained wide acceptance among the public and scientific community.²² Several natural compounds have been proven to show anti-inflammatory effects.

In particular, polyphenolic compounds have been given the most attention.²³ There is a paucity of information regarding the phytochemistry of the polyphenolic content and biological effects of P. khinjuk L. Therefore, the present study was designed to isolate and elucidate the structure of the main components of the aqueous methanol extract of P. khinjuk L. and to explore its potential anti-inflammatory effects in different experimental models.

Materials and Methods

Chemicals

Croton oil, hexadecyltrimethylammonium bromide, o-dianisidine, indomethacin, lipopolysaccharide (LPS) from Escherichia coli serotype O111:B4, modified Griess reagent, sodium nitrite, and gallic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA). Inflammatory-grade carrageenan was purchased from FMC (Rockland, ME, USA). The PGE₂ kit was purchased from R&D Systems Inc. (Minneapolis, MN, USA). The TNF-α kit was purchased from Assaypro LLC (St. Charles, MO, USA). Acetone, ethanol, ether, and pyridine were purchased from SDS (Peypin, France). The solvents methanol, water, and acetic acid of high-performance liquid chromatography (HPLC) grade were purchased from Merck Ltd. (Mumbai, India). Other chemicals and reagents used were of analytical grade.

Plant materials

P. khinjuk L. leaves were collected in April 2009 from the El-Zohreya Botanical Garden (Cairo, Egypt) and authenticated by Terease Labib, an agricultural engineer at El-Orman Botanical Garden (Giza, Egypt). A voucher specimen was deposited in the Herbarium of the Pharmacognosy Department, Faculty of Pharmacy, Ain Shams University, Cairo.

Instruments and materials for phytochemical investigation

Nuclear magnetic resonance

¹H and ¹³C nuclear magnetic resonance (NMR) spectra were obtained on a JOEL (Tokyo, Japan) ECA 500 spectrometer. ¹H chemical shifts were measured relative to the trimethylsilane scale by adding 39.5, and ¹³C chemical shifts were measured at 100 MHz, relative to dimethyl sulfoxide-d6 (DMSO-d6), and converted to trimethylsilane width equal to 6,000 Hz for ¹H and 22,000 Hz for ¹³C.

Ultraviolet

Ultraviolet (UV) spectroscopy was performed with a Shimadzu (Kyoto, Japan) UV/Vis spectrometer. Paper chromatography (PC) analysis was carried out on Whatman (Maidstone, UK) No. 1 paper, using the following solvent systems: (1) water; (2) 6% acetic acid; (3) n-butanol–acetic acid–water (BAW) (4:1:5 by volume, upper layer); and (4) n-butanol saturated with water. Solvents 2 and 3 were used for preparative PC (PPC) on Whatman No. 3MM paper. The Sephadex LH-20 (25–100 μm) column was from Sigma-Aldrich, and the polyamide 6 S for column chromatography was from Riedel-De Haen AG (Seelze-Hannover, Germany).

Plant extraction, isolation, and purification

Fresh leaves (2 kg) were exhaustively extracted with 10 L of aqueous methanol (75%). Leaves were boiled in double distilled water for exactly 2 hours. The aqueous extract was dried by lyophilization. The dry lyophilized extract was then further extracted with methanol (HPLC grade) for 30 minutes at 40°C to ensure complete extraction of phenolic components. This method helps to avoid extraction of other plant metabolites as carbohydrates, water-soluble protein, inorganics, lipids, or alkaloids, if any. Finally, the extract was completely evaporated in vacuo at low temperature until dryness to give 66 g of solid residue.

Two-dimensional PC of the extract proved the presence of a high percentage of phenolic constituents (green and blue color reaction with ferric chloride TS and ammonia).

Repeated fractionation of the extract (120 g) on polyamide 6S columns, using water followed by water–methanol mixtures of decreasing polarities, yielded eight fractions (I–VIII), which were individually subjected to further purification using subcolumns and PPC. Compounds 1 (35 mg), 2 (30 mg), 3 (20 mg), and 4 (24 mg) were obtained from Fraction II (eluted with 20% methanol) through subsequent PPC using BAW as a solvent. Compounds 5 (28 mg), 6 (30 mg), and 7 (20 mg) were obtained from Fraction VI (eluted with 60% methanol) through subsequent fractionation over column chromatography using Sephadex LH-20 and n-butanol saturated with water as a solvent system. Further purification was done using PPC using either 6% acetic acid as a solvent for compounds 5 and 6 or BAW as a solvent for compound 7.

Gallic acid (1) was obtained as an off-white amorphous powder (35 mg). Chromatographic analysis revealed an intense violet spot under UV and blue color with ferric chloride TS. The ratio of fronts (R _f) (×100) values were 72 in BAW, 56 in 6% acetic acid, and 44 in water. UV spectral data showed a λ_max (nm) in methanol of 272. ¹H-NMR spectral data gave δ (ppm) (DMSO-d6 at room temperature) of 6.89 (s, H-2 and H-6). ¹³C-NMR spectral data gave δ (ppm) (DMSO-d6) of 122.1 (C-1), 109.34 (C-2 and C-6), 138.3 (C-4), 145.9 (C-3 and C-5), and 168.8 (C-7). ¹H and ¹³C NMR data were identical to those reported in the literature.²⁴

3-O-Methyl gallate (4,5-dihydroxy-3-O-methylbenzoic acid) (2) was obtained as an off-white amorphous powder (30 mg) that appears as a violet spot under UV and blue color with ferric chloride TS. The R _f (×100) values were 77 in BAW, 50 in 6% acetic acid, and 50 in water. UV spectral data showed a λ_max (nm) in methanol of 277. ¹H-NMR spectral data gave δ (ppm) (DMSO-d6 at room temperature) of 6.89 (1H, s, H-2), 6.938 (1H, s, H-6), and 3.65 (3H, s, -OCH₃). ¹³C-NMR spectral data gave δ (ppm) (DMSO-d6) of 119.4 (C-1), 109 (C-2 and C-6), 145.3 (C-3), 146.2 (C-5), 139.5 (C-4), 166.9 (C-7, -COOH), and 52.1 (-OCH₃). ¹H and ¹³C NMR data were identical to those reported in the literature.²⁴

Quercetin-3-O-β-d-⁴C₁-galactopyranoside (hyperin) (3) was obtained as a yellow amorphous powder (20 mg), which appeared as a dark purple spot on PC under UV light, turning bright yellow by NH₃ vapor. The R _f (×100) was 54 in BAW, 30 in 6% acetic acid, and 20 in water. FeCl₃ reaction gave a green color. The UV absorption spectrum in methanol exhibited two major bands at λ_max of 269 and 363 nm. The structure of compound 3 was finally elucidated by ¹H-NMR spectroscopic analysis showed an aglycone moiety with δ (ppm) of 6.2 (1H, d, J=2.0 Hz, H-6), 6.3 (1H, d, J=2.0 Hz, H-8), 6.7 (1H, d, J=8.5 Hz, H-5'), 6.9 (1H, dd, J=8.5 and 2.5 Hz, H-6'), and 7.1 (1H, d, J=2.5 Hz, H-2') and a sugar moiety δ (ppm) of 5.2 (d, J=7.2 Hz, H-1 galactose) and 3.1–3.9 (m, sugar protons). ¹H-NMR data were identical to those reported in the literature.²⁵

Myricetin-3-O-α-l-¹C₄-rhamnopyranoside (myricitrin) (4) was obtained as a yellow powder (24 mg), which appeared as a dark purple spot on PC under UV light, turning yellow when fumed with NH₃. The R _f (×100) was 52 in BAW, 49 in 6% acetic acid, and 20 in water. Spraying with FeCl₃ gave a green color. The UV absorption spectrum in methanol exhibited two major bands at λ_max of 259 and 363 nm. ¹H-NMR spectral data showed an aglycone moiety δ (ppm) of 6.15 (1H, d, J=2.1 Hz, H-6), 6.33 (1H, d, J=2.1Hz, H-8), and 6.8 (2H, s, H-2', H-6') and a sugar moiety δ (ppm) of 5.1 (d, J=2.1 Hz, H-1''), 3.1–3.9 (m, sugar protons), and 0.79 (3H, d, J=6.2 Hz, H-6''). Compound 4 was finally identified by comparing these data with those reported for myricetin-3-O-α-l-¹C₄-rhamnopyranoside.²⁶

1,6-Digalloyl-β-d-glucose (5) was isolated as an off-white powder (28 mg). On PC, it appeared as a blue spot that turned bluish-violet upon spraying with 1% methanolic ferric chloride. The R _f (×100) was 58 in water, 64 in 6% acetic acid, and 43 in BAW. The UV spectrum showed absorbance at 277 nm. This was supported through complete acid hydrolysis of the parent compound to yield gallic acid and glucose as well as through partial acid hydrolysis, which led to the release of only one intermediate besides gallic acid. The intermediate was separated through PPC using BAW as solvent and then subjected to chromatographic and UV spectral analysis, which proved the identity as monogalloyl glucose. ¹H-NMR spectral data showed δ (ppm) (DMSO-d6 at room temperature) for the galloyl moiety of 6.82 (s) and 6.89 (s) and for the β-glucose moiety of 5.2 (1H, d, J=7.5 Hz, H-1), 4.99 (1H, t, J=7.5 Hz, H-2), and 3.6–4.0 (m, overlapping other sugar and hydroxyl proton signals). ¹H-NMR data were identical to those reported in the literature.²⁴

1,4-Digalloyl-β-d-glucopyranoside (6) was isolated as an off-white powder (30 mg). On PC, it appeared as a blue spot that turned bluish-violet upon spraying with 1% methanolic ferric chloride. The R _f (×100) was 60 in water, 65 in 6% acetic acid, and 48 in BAW. The UV spectrum showed absorbance at 276 nm. This was supported through complete acid hydrolysis of the parent compound to yield gallic acid and glucose as well as through partial acid hydrolysis, which led to the release of only one intermediate besides gallic acid. The intermediate was separated through PPC using BAW as solvent and then subjected to chromatographic and UV spectral analysis, which proved the identity as monogalloyl glucose. ¹H-NMR spectral data showed δ (ppm) (DMSO-d6 room temperature) for the galloyl moiety of 6.95 (s) and 7.1 (s) and for the β-glucose moiety of 5.3 (1H, d, J=7.5 Hz, H-1), 5.2 (1H, t, J=7.5 Hz, H-2), 5.41 (1H, t, J=7.5 Hz, H-3), and 3.3–4.2 (m, overlapping other sugar and hydroxyl proton signals). ¹³C-NMR spectral data showed δ (ppm) (DMSO-d6) for the β-glucose moiety of 94.1 (C-1), 71.1 (C-2), 73 (C-3), 68.3 (C-4), 73.9 (C-5), and 60.6 (C-6) and for the galloyl moiety of 120.48, 121.07 (C-1 in the two galloyl moieties), 109.22, 109.39, 109.64 (C-2 and C-6 in all galloyl moieties), 145.88, 146.04 (C-3 and C-5 in all galloyl moieties), 138.73, 138.88 (C-4 in all galloyl moieties), 166.03, and 166.4 (C=O carbons in all galloyl moieties). Compared with the literature, compound 6 was identified for the first time in this species.²⁷

Chromatographic conditions

A Shimadzu LC-8A HPLC system with a binary solvent delivery system (LC-8A), a column temperature controller (CTO-10AV), a Rheodyne (Rohnert Park, CA, USA) injector with a 20-μL sample loop, and a UV detector coupled with Class VP analytical software were used for analysis. A reverse-phase column (Phenomenex [Torrance, CA, USA] C-18, ODS-2; particle size, 5 μm; 250×4.6 mm) with an extended guard column was used as the stationary phase, and column temperature was maintained at 35°C. The mobile phase was a gradient elution of water containing 2% acetic acid (solvent A) and methanol (solvent B) at a flow rate of 1 mL/minute. The gradient program of solvent A in B (vol/vol) was as follows: 0–5 minutes, 100% A; 5–10 minutes, 90% A; 10–15 minutes, 90%; 15–20 minutes, 80% A; 20–25 minutes, 80%; 25–40 minutes, 40% A; and 40–60 minutes, 0% A. The chromatographic run was scanned at 280 nm.

Preparation of standard solution of gallic acid

Authentic standard gallic acid was dissolved in HPLC-grade methanol to prepare the calibration curve. Several dilutions in mobile phase were made. All standard solutions were filtered (pore size, 0.45 μm) and injected directly. The standard response curve was a linear regression fitted to triplicate values obtained at each of three concentrations (ranging from 0.002 to 0.004 mg/mL). The results, determined by the regression equation of the calibration curve (y=62.8x – 0.67, R ²=0.99), were expressed as milligrams of gallic acid equivalents per gram dry weight of raw material.

Preparation of plant extract for HPLC analysis

The dried methanolic extract of P. khinjuk L. leaves was dissolved in HPLC-grade methanol. The concentrations were determined by calculating the HPLC peak areas, which are proportional to the amount of analytes in a peak.

Animals

Throughout the experiments, adult male Sprague–Dawley rats weighing 150–175 g were obtained from the animal facility of King Fahd Medical Research Center, King Abdulaziz University, Jeddah, Saudi Arabia. Animals were housed at a temperature of 23±2°C with free access to water and standard food pellets. Rats were acclimatized in our animal facility for at least 1 week prior to any experiment. Procedures involving animals and their care were conducted in conformity with the institutional guidelines of King Abdulaziz University.

Experimental protocol

The potential anti-inflammatory activity of P. khinjuk L. extract was examined in three experimental models: the carageenen-induced rat edema model, the croton oil–induced ear edema model, and the rat air pouch model. Collectively, the parameters assessed included paw volume, PGE₂ level, ear edema, tissue MPO, histopathology, NO, and TNF-α. The control group animals received the same experimental handling as those of the test groups except that the drug treatment was replaced with appropriate volumes of the dosing vehicle. Indomethacin, a potent NSAID, was suspended in 0.5% carboxymethylcellulose and used as a reference drug. It was administered in doses of 10 mg/kg in rat paw edema and ear edema experiments, respectively. The choice of the doses used and times of measurement and sampling was based on pilot studies in our laboratory.

Measurement of paw volume and PGE₂ in carrageenan-induced rat edema model

Twenty-four rats were equally divided into four groups (I–IV). Animals were fasted, with free access to water, 16 hours before the experiment. Using an intragastric tube, Groups I and II were given the vehicle (0.5% carboxymethylcellulose), whereas Group III was treated with P. khinjuk L. extract. Animals in Group IV received indomethacin as a standard anti-inflammatory drug. The dosing volume was kept constant (10 mL/kg) for all the orally treated groups. Thirty minutes after oral treatment, Group I received 0.05 mL of saline, whereas Groups II–IV received 0.05 mL of carrageenan (1% solution in saline) subcutaneously on the plantar surface of the right hind paw. The right hind paw volume was measured immediately after carrageenan injection by water displacement using a plethysmometer (model 7140, Ugo Basile, Comerio, Italy).²⁸ The paw volume was remeasured 3 hours after carrageenan injection and immediately before decapitation.

The level of PGE₂ was then assessed. Right hind paws were removed. A volume of 0.1 mL of saline containing 10 μM indomethacin was injected to aid removal of the eicosanoid-containing fluid and to stop further production of PGE₂. Paws were incised with a scalpel and suspended off the bottom of polypropylene tubes with Eppendorf pipette tips to facilitate drainage of the inflammatory exudates. For the purpose of the removal of the inflammatory exudates, paws were centrifuged at 1800 g for 15 minutes.²⁹ PGE₂ was quantified in the collected exudates using a PGE₂ enzyme-linked immunosorbent assay kit. This assay is based on the forward sequential competitive binding technique in which PGE₂ in a sample competes with horseradish peroxidase–labeled PGE₂ for a limited number of binding sites on monoclonal antibodies. PGE₂ in the sample is allowed to bind to the antibody in the first incubation. During the second incubation, horseradish peroxidase–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.³⁰

Assessment of ear edema, tissue MPO activity, and histopathology in croton oil–induced ear edema model in rats

The experiment was performed using a slight modification of the procedure described by Tonelli et al.³¹ An irritant solution was prepared by dissolving 4 parts croton oil (the irritant) in a solvent mixture of 10 parts ethanol, 20 parts pyridine, and 66 parts ethyl ether. The P. khinjuk L. extract and indomethacin were dissolved in the same vehicle of the irritant. Twenty-four rats were equally divided into four groups (I–IV). The irritant solution was applied in a volume of 20 μL topically on both sides of the right ear; the left ear was kept untreated to serve as a control. Group I served as the negative control and hence received only the irritant-free solvent mixture, whereas Group II received the croton oil solution. The third and fourth groups received the P. khinjuk L. extract (100 mg/kg orally) and indomethacin (10 mg/kg orally), respectively. One hour later, the solvent mixtures were re-administered to the groups. After 4 hours, animals were sacrificed. An 8-mm cork borer was used to punch out discs from both the treated and the control ears. The two punches were weighed immediately after decapitation, and the difference in weight was used to assess the inflammatory response.

The entire tissue of the right ear was homogenized for 10 minutes in an ice bath (10%, wt/vol) in 50 mM phosphate buffer (pH 6.0) containing 0.5% hexadecyltrimethylammonium bromide with a IKA^® (Staufen, Germany) homogenizer. Tissue suspensions were centrifuged at 40,000 g for 15 minutes. An aliquot of 0.1 mL of the supernatant was added to 2.9 mL of 50 mM phosphate buffer (pH 6.0) containing 0.167 mg/mL o-dianisidine dihydrochloride, serving as the MPO substrate, and 0.0005% hydrogen peroxide. The change in absorbance was measured at 460 nm with a Shimadzu UV-1601 spectrophotometer at 25°C. MPO activity was quantified kinetically; change in absorbance was measured over a period of 2 minutes, sampled at intervals of 15 seconds.³² The maximal change in absorbency per minute was used to calculate the units of MPO activity based on the molar absorbency index of oxidized o-dianisidine dihydrochloride, which equals 1.13×10⁴ M ⁻¹ cm⁻¹. One unit of MPO is defined as that degrading 1 μmol of peroxide/minute at 25°C. Results were expressed as units of activity per milligram of protein. Protein content was determined according to the method of Lowry et al.³³

A representative ear tissue from each group was fixed in 10% formol saline for 24 hours. Washing was done in tap water, and then serial dilutions of alcohol (methyl, ethyl, and absolute ethyl) were used for dehydration. Specimens were cleared in xylene and embedded in paraffin at 56°C in a hot air oven for 24 hours. Paraffin beeswax tissue blocks were prepared for sectioning at 4 μm thickness with a sledge microtome. The tissue sections obtained were collected on glass slides, deparaffinized, and stained by hematoxylin and eosin, and examination was done with the light microscope.³⁴

Measurement of NO and TNF-α in the rat air pouch model

Twenty-four rats uniformly divided in four groups were used in the study. Air pouches were formed by subcutaneous injection of 20 mL of sterile air in the suprascapular area of the back of the animal. Three days later, the pouches were re-inflated with 10 mL of sterile air. After another 3 days, a 100 μg/mL solution of LPS in physiological saline (1 mL/kg) was administered intrapouch to Groups II–IV; the control group (Group I) received physiological saline (1 mL/kg) intrapouch. Thirty minutes later, drugs were injected, also intrapouch. The volume of administered solutions was kept constant at 3 mL. Groups I and II were administered only saline, Group III received the P. khinjuk L. extract (100 mg/kg), and Group IV received indomethacin (10 mg/kg). Eight hours later animals were sacrificed.³⁵ Each pouch was lavaged using 1 mL of sterile physiological saline. The lavage fluid was then centrifuged at 3,000 g for 5 minutes. Supernatant was used immediately for analysis of NO and TNF-α.

NO assay is based on measuring total nitrite/nitrate level based on reduction of any nitrate to nitrite by vanadium trichloride followed by detection of total nitrite by modified Griess reaction [sulfanilamide and N-(1-naphthyl)-ethylenediamine]. A chromophore is formed by diazotization of sulfanilamide with acidic nitrite followed by coupling with the bicyclic amine N-(1-naphthyl)-ethylenediamine.³⁶ In brief, 250 μL of vanadium trichloride solution was added to 250 μL of air pouch exudates followed by rapid addition of 250 μL of modified Griess reagent. The mixture was incubated at 37°C for 30 minutes and then cooled. Absorbance was measured at 540 nm using a Shimadzu UV-1601 spectrophotometer against a blank treated in a similar manner to the test solution but using 250 μL of double distilled water instead of sample. The concentration of NO was expressed as micromolar using a standard curve.

The assay of TNF-α depends on a quantitative sandwich enzyme immunoassay technique that measures TNF-α in 4.5 hours. A murine monoclonal antibody specific for rat TNF-α was precoated onto a microplate. TNF-α in standards and samples was sandwiched by the immobilized antibody and a biotinylated polyclonal antibody specific for rat TNF-α, which is recognized by a streptavidin–peroxidase conjugate. All unbound material was then washed away, and a peroxidase enzyme substrate was added. The color development was stopped, and the intensity of the color was measured at 450 nm using a microplate reader.³⁰

Statistical analysis

Data are presented as mean±SD values. Statistical analysis was performed using one-way analysis of variance followed by Tukey–Kramer multiple comparisons tests. The .05 level of probability was used as the criterion for significance. All statistical analyses were performed using GraphPad (La Jolla, CA, USA) InStat software version 3. Graphs were sketched using GraphPad Prism software version 4 (ISI^® Software, La Jolla, CA, USA).

Results

Phytochemical investigation of P. khinjuk L.

Phytochemical investigation of the aqueous methanol extract indicated the presence of phenolic metabolites. Following column chromatographic fractionation of the P. khinjuk L. conventional and spectral analysis mainly by UV, ¹H and ¹³C NMR spectroscopy was applied. As shown in Figure 1, the isolated compounds were gallic acid (1), methyl gallate (2), quercetin-3-O-β-d-⁴C₁-galactopyranoside (hyperin) (3), myricetin-3-O-α-l-¹C₄-rhamnopyranoside (myricitrin) (4), 1,6-digalloyl-β-d-glucose (5), 1,4-digalloyl-β-d-glucopyranoside (6), and 2,3-di-O-galloyl-(α/β)-⁴C₁-glucopyranose (nilocitin) (7). Compound 6 was isolated and identified for the first time from the genus Pistachia.

FIG. 1.

Phenolic compounds isolated from the aqueous methanol extract of P. khinjuk L.: gallic acid (1), methyl gallate (2), quercetin-3-O-β-d-⁴C₁-galactopyranoside (hyperin) (3), myricetin-3-O-α-l-¹C₄-rhamnopyranoside (myricitrin) (4), 1,6-digalloyl-β-d-glucose (5), 1,4-digalloyl-β-d glucopyranoside (6), and 2,3-di-O-galloyl-(α/β)-⁴C₁-glucopyranose (nilocitin) (7).

HPLC investigation of P. khinjuk L.

Reversed-phase HPLC coupled with UV/Vis was used at 280 nm to identify and quantify phenolic compounds in P. khinjuk L. The concentrations were determined by calculating the HPLC peak areas, which are proportional to the amount of analytes in a peak, and are presented as the mean value of two determinations, which were highly reproducible. Gallic acid has been identified in P. khinjuk L. extract according to the retention time and spectral characteristics of its peak against that of the standard gallic acid. The methanol extract of P. khinjuk L. contained approximately 81% gallic acid.

Effect of P. khinjuk L. extract on paw volume and PGE₂ in carrageenan-induced rat edema

Intraplantar injection of carrageenan to rats resulted in severe discernible inflammation and significant increase in the mean volume of the challenged paw compared with that of the untreated paws (137.3% of the untreated paws) (Table 1). Pretreatment of rats with P. khinjuk L. extract at a dose of 100 mg/kg significantly inhibited the carrageenan-induced increase in the edema volume of the paws by 46.8%. Indomethacin inhibited the induced edema by 80.8%.

Table 1.

Effect of P. khinjuk L. Extract on Paw Volume in Carrageenan-Induced Rat Edema

Group	Paw volume (mL)	Edema inhibition (%)
I (control)	1.26±0.032	—
II (carrageenan)	1.73±0.033^a	—
III (carrageenan + extract)	1.51±0.1^{a b}	46.8%
IV (carrageenan + indomethacin)	1.35±0.035^b	80.8%

Statistically significant difference from the control group (Group I) at P<.05.

Statistically significant difference from the carrageenan-induced group (Group II) at P<.05.

Carrageenan challenge resulted in a >10-fold increase in PGE₂ concentration in inflammatory exudates in Group II compared with unchallenged animals in Group I (Fig. 2). Animals receiving P. khinjuk L. extract showed significant reduction of the PGE₂ concentration in exudates by 87.45% of the carrageenan-treated animals. The standard indomethacin lowered PGE₂ level in carrageenan-challenged animals, to values approaching normal levels.

FIG. 2.

Effect of P. khinjuk L. extract on prostaglandin E₂ (PGE₂) production in exudates from carrageenan-treated rats. Statistically significant (P<.05) differences are indicated: ^afrom the control group, ^bfrom the carrageenan-induced group.

Effect of P. khinjuk L. extract on croton oil–induced ear edema, MPO tissue activity, and histopathological changes

Application of croton oil to rat ears caused a massive increase in the weight of the ear punch from control animals by 83.4%. Pretreatment of rats with P. khinjuk L. extract significantly reduced the increase of punch weight from unchallenged ears to about 52.4%. Indomethacin pretreatment produced significant reduction of punch weight from control values to 37.4%, as shown in Table 2.

Table 2.

Effect of P. khinjuk L. Extract on Croton Oil–Induced Rat Ear Edema

		Percentage punch weight
Group	Ear punch weight (g)	Increase from control	Reduction from Group II
I (control)	0.0187±0.0008	—	—
II (croton oil)	0.0343±0.0008^a	83.4%	—
III (croton oil + extract)	0.0285±0.0005^{a b}	52.4%	−16%
IV (croton + indomethacin)	0.0257±0.0009^{a b}	37.4%	−25%

Statistically significant difference from the control group at P<.05.

Statistically significant difference from the croton oil–induced group at P<.05.

Assay of MPO activity in rat ears indicated that application of croton oil increased the enzyme activity by more than 25-fold. Pretreatment with P. khinjuk L. extract at a dose of 100 mg/kg, however, reduced MPO activity by 57.53% of the induced ear. Indomethacin pretreatment resulted in significant protection against croton oil–induced enhancement of MPO activity (Fig. 3).

FIG. 3.

Effect of P. khinjuk L. extract on myeloperoxidase (MPO) activity in croton oil-induced edema. Statistically significant (P<.05) differences are indicated: ^afrom the control group, ^bfrom the croton oil–induced group.

Histopathological examination of the ear tissue confirmed those results obtained by assessing MPO activity. Figure 4A shows normal histological structure of the epidermal and dermal layers as well as underlying cartilage with no obvious neutrophil infiltration. The croton oil–treated rats showed massive neutrophil infiltration in the dermal layer (Fig. 4B). Figure 4C, which represents ear tissue treated with 100 mg/kg P. khinjuk L. extract, shows less cellular infiltration in the dermal layer compared with the croton oil–treated group. Figure 4D, corresponding to indomethacin treatment, shows almost intact dermal and cartilaginous structures with little neutrophil infiltration.

FIG. 4.

Effect of P. khinjuk L. extract on histopathological changes in the croton oil–induced ear edema experiment. Magnification×64. (A) Normal histological structure of the epidermal and dermal layers as well as underlying cartilage with no obvious neutrophil infiltration. (B) Croton oil–treated rats with massive neutrophil infiltration in the dermal layer. (C) Ear tissue treated with 100 mg/kg P. khinjuk L. extract shows less cellular infiltration in the dermal layer compared with the croton oil–treated group. (D) Ear tissue treated with indomethacin shows almost intact dermal and cartilaginous structures with little neutrophil infiltration. a, adipose tissue; c, cartilage; d, dermis; e, epidermis; h, hair follicle.

Effect of P. khinjuk L. extract on NO and TNF-α in the rat air pouch model

Injection of LPS into the air pouch caused about a 10-fold increase of NO production compared with that untreated animals (Group I). Injection of P. khinjuk L. extract at 100 mg/kg significantly reduced the NO level by 81.14%, compared with Group II (Fig. 5). Similarly, indomethacin treatment significantly lowered the NO level by 85.3%.

FIG. 5.

Effect of P. khinjuk L. extract on nitric oxide production in the rat air pouch model. Statistically significant (P<.05) differences are indicated: ^afrom the control group, ^bfrom the lipopolysaccharide (LPS)-induced group.

In addition, LPS injection caused about a fourfold increase in TNF-α level in the pouch exudates compared with the untreated group. Treatment with P. khinjuk L. extract at a dose of 100 mg/kg lowered TNF-α release by 32.62%, compared with the untreated group (Fig. 6). Indomethacin could lower TNF-α level in LPS-challenged animals, approaching normal levels.

FIG. 6.

Effect of P. khinjuk L. extract on tumor necrosis factor-α (TNF-α) release in the rat air pouch model. Statistically significant (P<.05) differences are indicated: ^afrom the control group, ^bfrom the lipopolysaccharide (LPS)-induced group.

Discussion

Literature review of the genus Pistacia had shown its richness in polyphenolics.³⁷ Plants containing polyphenolic compounds had shown their efficacy in many pharmacological activities, as anti-inflammatory,³⁸ antioxidant, hepatoprotective, heart disease protective, and anticancer activities.³⁹ Therefore, the aim of the present study was to isolate and elucidate the structure of the main components in an aqueous methanol extract of P. khinjuk L. and to evaluate its potential anti-inflammatory effects. In the present study, evaluation of anti-inflammatory effects was undertaken using different animal models to fully investigate the potential of P. khinjuk L. for treatment of inflammatory disorders. Phytochemical investigation of the aqueous methanol extract indicated the presence of phenolic metabolites. These metabolites were gallic acid (1), methyl gallate (2), quercetin-3-O-β-d-⁴C₁-galactopyranoside (hyperin) (3), myricetin-3-O-α-l-¹C₄-rhamnopyranoside (myricitrin) (4), 1,6-digalloyl-β-d-glucose (5), 1,4-digalloyl-β-d-glucopyranoside (6), and 2,3-di-O-galloyl-(α/β)-⁴C₁-glucopyranose (nilocitin) (7). It is noteworthy that compound 6 was isolated and identified for the first time from the genus Pistachia.

Acute inflammation such as carrageenan-induced paw edema involves the synthesis or release of mediators at the injured site. These mediators include PGs (especially the E series), histamine, bradykinins, leukotrienes, and serotonin and cause fever and pain.⁴⁰ Carrageenan-induced paw edema is an experimental animal model for acute inflammation, and it is believed to be biphasic: the early phase (1–2 hours) of this assay is mainly mediated by histamine, serotonin, and an increasing synthesis of PGs in the damaged tissue surroundings; the late phase is sustained by PG release.⁴¹

In the present study, the rat paw edema model showed the ability of P. khinjuk L. extract to significantly reduce paw edema, indicating its potential anti-inflammatory activity. Such a conclusion was further substantiated by the ability of P. khinjuk L. extract to significantly reduce the PGE₂ level in the inflammatory exudates in the same model. The results of croton oil–induced ear edema support this finding. A reduction in ear punch weight was shown after pretreatment with P. khinjuk L. extract compared with the croton oil–alone condition. This activity might be justified by the adequate distribution of P. khinjuk L. extract to ear tissues. The findings of the same experiment demonstrated that P. khinjuk L. extract reduced MPO activity in challenged ear tissue. This could be explained based on inhibition of neutrophil infiltration, which was confirmed visually by microscopic examination of the ear tissue. It is noteworthy that the effectiveness of the extract in inhibiting neutophil infiltration was comparable to that of indomethacin. This might imply the potential utility of natural products as substitutes for NSAIDs. These findings are in line with the plant's content of polyphenolics and are supported by the work of Paula et al.,⁴² who reported that other herbs such as Aegiceras corniculatum rich in polyphenols were able to inhibit the production of eicosanoids in rat neutrophils. Cutaneous inflammation is characterized by the infiltration of polymorphonuclear leukocytes such as neutrophils.⁴³ MPO is a major component of azurophilic granules of neutrophils. Oxygen-dependent microbicidal activity requires MPO as the critical enzyme for the generation of hypochlorous acid and other toxic oxygen products.⁴⁴ The primary function of neutrophils is the release of MPO into the phagosome containing the ingested microorganism, leading to a rapid microbicidal effect. MPO can be released to the outside of the cell, inducing damage to adjacent tissue and thus contributing to the pathogenesis of inflammation.⁴⁵

NO is a relatively stable free radical, having numerous promiscuous roles.⁴⁶ NO synthesis can be greatly amplified by LPS.⁴⁷ Several studies have demonstrated that inflammation correlates with the level of NO.⁴⁸ In the current study, the extract of P. khinjuk L. has shown remarkable reduction of nitrite accumulation in the air pouch model. This finding could be attributed to the high content of quercetin flavonoids and phenolic metabolites in the P. khinjuk L. extract observed in the current study. These have been previously shown to possess potent antioxidant and anti-inflammatory activity.⁴⁹ Furthermore, the lowering of NO level after P. khinjuk L. extract administration may account for the reduction in PGE₂ level because many reports have shown that NO rapidly and strongly stimulates cyclooxygenase enzymes.⁵⁰ In addition, NO synthase inhibitors reduce PGE₂ biosynthesis.⁵¹

TNF-α is a pleiotropic cytokine that plays a critical role in both acute and chronic inflammation.⁵² TNF-α promotes an acute-phase reaction.⁵³ Several inflammagens have the ability to induce TNF-α synthesis. The formation of several small molecular mediators of inflammation is linked with TNF-α and thus contributes to the range of mediators that critically control inflammation.⁵⁴ TNF-α facilitates inflammatory cell infiltration by promoting the adhesion of neutrophils and lymphocytes to endothelial cells.⁵⁵ When the TNF-α effect is specifically blocked, the severity of inflammation is reduced.⁵⁶ More important is that TNF-α induces the synthesis of PGE₂.⁵³ P. khinjuk L. extract significantly inhibited TNF-α release, which is in agreement with the PGE₂ reduction obtained in the current study. The observed anti-inflammatory activity of P. khinjuk L. aqueous methanol extract could be explained by our finding that the extract is rich in flavonoids and phenolic metabolites, represented by the high yield of gallic acid and galloylated sugars.³⁸

In conclusion, the isolation and NMR spectral data of compound 6 from the genus Pistacia are revealed in the current study for the first time. Also, P. khinjuk L. aqueous methanol extract possesses anti-inflammatory activity in several animal models. This is evidenced by decreased paw edema, PGE₂ content in exudates, ear edema, MPO activity, NO generation, and TNF-α formation as well as amelioration of inflammatory histopathological changes.

Footnotes

Acknowledgments

The authors would like to thank Prof. Adel B. Kholoussy, Professor of Pathology, Cairo University, for his help in the histopathological examinations.

Author Disclosure Statement

No competing financial interests exist.

References

Zhao

, Sun

, Hou

, Zhao

, Wei

, Xin

. Antioxidant properties of two gallotannins isolated from the leaves of Pistacia weinmannifolia. Biochim Biophys Acta, 2005; 1725:103–110.

Hou

, Peng

, Liu

, Lin

, Sun

. Gallotannins and related polyphenols from Pistacia weinmannifolia. Planta Med, 2000; 66:624–626.

Topçu

, Mehmet

, Bilici

, Sankürkcü

, Öztürk

, Ulubelen

. A new flavone from antioxidant extracts of Pistacia terebinthus. Food Chem Toxicol, 2007; 103:816–822.

Yalpani

, Tyman

JHP

. The phenolic acids of Pistachia vera. Phytochemistry, 1983; 22:2263–2266.

Ansari

, Ali

, Qadry

, Siddiqui

. Analgesic activity of tetracyclic triterpenoids isolated from Pistacia integerrima galls. Update Ayurveda, 1994; 94:73.

Ansari

, Ali

, Qadry

. New tetracyclic triterpenoids from Pistacia integerrima galls. Pharmazie, 1994; 49:356–357.

Longo

, Scardino

, Vasapollo

. Identification and quantification of anthocyanins in the berries of Pistacia lentiscus L., Phillyrea latifolia L. and Rubia peregrina L. Innov Food Sci Emerg Technol, 2007; 8:360–364.

Liu

, An Geng

, Liu

. A new pyrrolidone derivative from Pistacia chinensis. Chin Chem Lett, 2008; 19:65–67.

Özcan

, Tzakou

, Couladis

. Essential oil composition of the turpentine tree (Pistacia terebinthus L.) fruits growing wild in Turkey. Food Chem, 2009; 114:282–285.

10.

Mahmoudi

, Ebrahimzadeh

, Nabavi

, Hafezi

, Nabavi

, Eslami

. Antiinflammatory and antioxidant activities of gum mastic. Eur Rev Med Pharmacol Sci, 2010; 14:765–769.

11.

Ahmad

, Waheed

, Farman

, Qayyum

. Analgesic and anti-inflammatory effects of Pistachia integerrima extracts in mice. J Ethnopharmacol, 2010; 129:250–253.

12.

Gioxari

, Kaliora

, Papalois

, Agrogiannis

, Triantafillidis

, Andrikopoulos

. Pistacia lentiscus resin regulates intestinal damage, inflammation in trinitrobenzene sulfonic acid-induced colitis. J Med Food Epub, 2011 May 25 doi:10.1089/jmf.2010.0240.

13.

Walport

, Duff

. Cells and mediators of inflammation. Oxford Textbook of Rheumatology. Maddison

, Isenberg

, Woo

, Glass

. Oxford University Press: Oxford, UK, 1993; 285–295.

14.

Majno

, Joris

. Cells, Tissues, Disease: Principles of General Pathology. Blackwell Science: Cambridge, MA, 1996.

15.

Helliwell

, Adams

, Mitchell

. Prostaglandin synthases: recent developments and a novel hypothesis. Prostaglandins Leukot Essent Fatty Acids, 2004; 70:101–113.

16.

Goulet

, Snouweart

, Latour

, Coffman

. Altered inflammatory responses in leukotriene-deficient mice. Proc Natl Acad Sci U S A, 1994; 91:12852–12856.

17.

Amin

, Vyas

, Attur

, Leszczynsca-Piziak

. The modes of action of aspirin-like drugs: effect on inducible nitric oxide synthase. Proc Natl Acad Sci U S A, 1995; 92:7926–7930.

18.

Marucha

, Zeff

, Kreutzer

. Cytokine-induced IL-1β gene expression in the human polymorphonuclear leukocyte: transcriptional and post-transcriptional regulation by tumor necrosis factor and IL-1. J Immunol, 1991; 147:2603–2608.

19.

Miller

. Protective effects of prostaglandins against gatric mocosal damage: current knowledge and proposed mechanisms. Am J Physiol, 1983; 245:G601–G623.

20.

Robert

, Hanchar

, Lancaster

, Nezamis

. Prostacyclin inhibits enteropooling and diarrhea. Prostacyclin. Vane

, Bergstrom

. Raven Press: New York, 1979; 147–158.

21.

Wallace

, Bak

, McKnight

, Asfaha

, Sharkey

, MacNaughton

. Cyclooxygenase-1 contributes to inflammatory responses in rats and mice: implications for gastrointestinal toxicity. Gastroenterology, 1998; 115:101–109.

22.

Bauer

. Herbal therapy: what a clinician needs to know to counsel patients effectively. Mayo Clin Proc, 2000; 75:835–841.

23.

Kupeli

, Sahin

, Calis

, Yesilada

, Ezer

. Phenolic compounds of Sideritis ozturkii and their in vivo anti-inflammatory and antinociceptive activities. J Ethnopharmacol, 2007; 112:356–360.

24.

Nawwar

MAM

, Hussein

SAM

, Merfort

. NMR spectral analysis of polyphenols from Punica granatum. Phytochemistry, 1994; 36:793–798.

25.

Mahmoud

, Moharam

, Marzouk

, Linscheid

, Saleh

. Polyphenolic constituents of Callistemon lanceolatus leaves. Pharmazie, 2002; 57:494–496.

26.

Fossen

, Larsen

, Kiremire

, Andersen

. Flavonoids from blue flowers of Nymphae caerulea. Phytochemistry, 1999; 51:1133–1137.

27.

Lee

, Tanaka

, Nonaka

, Nishioka

. Tannins and related compounds. Part 115. Dimeric ellagitannins from Alnus japonica. Phytochemistry, 1992; 31:2835–2839.

28.

Winter

, Risley

, Nuss

. Carrageenin-induced edema in hind paw of the rat as an assay for anti-inflammatory drugs. Proc Soc Exp Biol Med, 1962; 111:544–547.

29.

Mnich

, Veenhuizen

, Monahan

et al. Characterization of a monoclonal antibody that neutralizes the activity of prostaglandin E2. J Immunol, 1995; 155:4437–4444.

30.

Virella

. Immunoserology. Medical Immunology. Virella

. Marcel Dekker: New York, 1998; 259–281.

31.

Tonelli

, Thibault

, Ringler

. A bioassay for the concomitant assessment of the antiphlogistic and thymolytic activities of topically applied steroids. Endocrinology, 1965; 77:625–630.

32.

Bradely

, Priebat

, Christensen

, Rothstein

. Measurement of cutaneous inflammation: estimation of neutrophil content with an enzyme marker. J Invest Dermatol, 1982; 78:206–209.

33.

Lowry

, Rosebrough

, Farr

, Randall

. Protein measurement with the Folin phenol reagent. J Biol Chem, 1951; 193:265–275.

34.

Bancroft

, Stevens

, Turner

. Theory, Practice of Histological Techniques. Churchill Livingstone: New York, 1996.

35.

Edwards

JCW

, Sedgwick

, Willoughby

. The formation of a structure with the features of synovial lining by subcutaneous injection of air: an in vivo tissue culture system. J Pathol, 1981; 134:147–156.

36.

Miranda

, Espey

, Wink

. A rapid, simple spectrophotometric method for simultaneous detection of nitrate and nitrite. Nitric Oxide, 2001; 5:62–71.

37.

Kawashty

, Mosharrafa

SAM

, El-Gibali

, Saleh

NAM

. The flavonoids of four Pistacia species in Egypt. Biochem System Ecol, 2000; 28:915–917.

38.

Kroes

, Van den Berg

AJJ

, Ufford

HCQ

, Van

Dijk H

, Labadie

. Anti-inflammatory activity of gallic acid. Planta Med, 1992; 58:499–504.

39.

Dahanukar

, Kulkarni

, Rege

. Pharmacology of medicinal plants. Indian J Pharmacol, 2000; 32:S81–S118.

40.

Silbernagl

, Lang

. Atlas de Poche de Physiopathologie. Flammarion: Paris, 2000.

41.

Brito

ARMS

, Antonio

. Oral anti-inflammatory and antiulcerogenic activities of a hydroalcoholic extract and partitioned fractions of Turnera ulmifolia (Turneraceae) J Ethnopharmacol, 1998; 61:215–228.

42.

Paula

, Kabeya

, Kanashiro

et al. Modulation of human neutrophil oxidative metabolism and degranulation by extract of Tamarindus indica L. fruit pulp. Food Chem Toxicol, 2009; 47:163–170.

43.

Lawley

, Kubota

. Cell adhesion molecules and cutaneous inflammation. Semin Dermatol, 1991; 10:256–259.

44.

Nauseef

, Olsson

, Arnljots

. Biosynthesis and processing of myeloperoxidase—a marker for myeloid cell differentiation. Eur J Haematol, 1988; 40:97–110.

45.

Klebanoff

. Myeloperoxidase. Proc Assoc Am Physicians, 1999; 111:383–389.

46.

Moncada

, Palmer

RMJ

, Higgs

. Nitric oxide: physiology, pathophysiology and pharmacology. Pharmacol Rev, 1991; 43:109–142.

47.

Kou

, Schroeder

. The emerging multifaceted roles of nitric oxide. Ann Surg, 1995; 221:220–235.

48.

Miller

MJS

, Grisham

. Nitric oxide as a mediator of inflammation? You had better believe it. Mediat Inflamm, 1995; 4:387–396.

49.

Conforti

, Sosa

, Marrelli

et al. In vivo anti-inflammatory and in vitro antioxidant activities of Mediterranean dietary plants. J Ethnopharmacol, 2008; 116:144–151.

50.

Sautebin

, Ialenti

, Ianaro

, Di Rosa

. Modulation by nitric oxide of prostaglandin biosynthesis in the rat. Br J Pharmacol, 1995; 114:323–328.

51.

Salvemini

, Settle

, Masferrer

, Seibert

, Currie

, Needleman

. Regulation of prostaglandin production by nitric oxide: an in vivo analysis. Br J Pharmacol, 1995; 114:1171–1178.

52.

Holtmann

, Schuchmann

, Zeller

, Galle

, Neurath

. The emerging distinct role of TNF-receptor 2 (p80) signaling in chronic inflammatory disorders. Arch Immunol Ther Exp (Warsz), 2002; 50:279–288.

53.

Arai

, Lee

, Miyajima

, Miyatake

, Arai

, Yokota

. Cytokines: coordinators of immune and inflammatory responses. Annu Rev Biochem, 1990; 59:783–836.

54.

Harada

, Sekido

, Akahoshi

, Wada

, Mukaida

, Matsushima

. Essential involvement of interleukin-8 (IL-8) in acute inflammation. J Leukoc Biol, 1994; 56:559–564.

55.

Gamble

, Harlan

, Klebanoff

, Vadas

. Stimulation of the adherence of neutrophils to umbilical vein endothelium by human recombinant tumor necrosis factor. Proc Natl Acad Sci U S A, 1985; 82:8667–8671.

56.

Dinarello

. Proinflammatory and anti-inflammatory cytokines as mediators in the pathogenesis of septic shock. Chest, 1997; 112,Suppl 6:321S–329S.