Investigation into the mechanism of action of essential oil of Pistacia integerrima for its antiasthmatic activity

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Investigation into the mechanism of action of essential oil of Pistacia integerrima for its antiasthmatic activity Q1

R.L. Shirole a, A.A. Kshatriya a, R. Kulkarni b, N.L. Shirole c, M.N. Saraf a,n a

Department of Pharmacology, Bombay College of Pharmacy, Kalina, Santacruz (E), Mumbai, India Department of Clinical Pharmacology T. N. Medical College and B. Y. L. Nair Charitable Hospital, Mumbai, India c Department of Pharmaceutical Chemistry, A. R. A. College of Pharmacy, Nagaon, Dhule, India b

art ic l e i nf o Article history: Received 7 July 2013 Received in revised form 30 December 2013 Accepted 7 February 2014

Keywords: Pistacia integerrima J.L. Stewart ex Brandis Asthma Q3 Lipopolysaccharide Essential oil Spirometry

a b s t r a c t Q2 Ethnopharmacological relevance: Pistacia integerrima J.L. Stewart ex Brandis locally known as Karkatashringi is an important medicinal plant whose galls are valued in traditional medicine used in India for the treatment of asthma, chronic bronchitis, phthisis, diarrhea, fever, other ailments for the respiratory tract, and as antispasmodic, carminative, antiamoebic and anthelmintic. However, in vitro and in vivo investigations providing new insights into its pharmacological properties have not been thoroughly investigated yet. The present investigation aimed to elucidate the probable mechanism of antiasthmatic action of essential oil of Pistacia integerrima J.L. Stewart ex Brandis galls (EOPI). Methods: EOPI was tested using in vitro studies such as antioxidant activity, mast cell degranulation, angiogenesis, isolated guinea pig ileum preparation and soyabean lipoxidase enzyme activity. In vivo studies included lipopolysaccharide-induced bronchial inflammation in rats and airway hyperresponsiveness in ovalbumin in sensitized guinea pigs using spirometry. Results: EOPI (5–30 mg/ml) inhibits 5-lipoxidase enzyme activity with IC50 of 19.71 mg/ml and DPPH scavenging activity up to 100 mg/ml with maximum inhibition of 44.9372.53% at 100 mg/ml. Pre-treatment with EOPI inhibited erythropoietin-induced angiogenesis. It showed dose dependent (10, 30 and 100 mg/ml) anti-allergic activity by inhibiting compound 48/80 induced mast cell degranulation to an extent 19.0870.47%. The finding that essential oil induced inhibition of transient contraction of acetylcholine in calcium free medium, and relaxation of S-(-)-Bay 8644-precontracted isolated guinea pig ileum jointly suggests that the L-subtype Cav channel is involved in spasmolytic action of EOPI. Treatment with EOPI dose dependently (7.5, 15 and 30 mg/kg i.p.) inhibited lipopolysaccharide-induced increase in total cell count, neutrophil count, nitrate–nitrite, total protein, albumin levels in bronchoalveolar fluid and myeloperoxidase levels in lung homogenates. Roflumilast was used as a standard. EOPI reduced the respiratory flow due to gasping in ovalbumin sensitized guinea pigs. Conclusion: The study demonstrates the effectiveness of essential oil of Pistacia integerrima J.L. Stewart ex Brandis galls in bronchial asthma possibly related to its ability to inhibit L-subtype Cav channel, mast cell stabilization, antioxidant, angiostatic and through inhibition of 5-lipoxygenase enzyme. & 2014 Published by Elsevier Ireland Ltd.

Abbreviations:: %, Percentage; 1C, Degree celcius; m, microgram; ml, microliter; A, Alveoli; ACh, Acetylcholine; AHR, Airway Hperresponsiveness; ANOVA, Analysis of Variance; AS, Inter Alveolar Septum; BAL, Bronchoalveolar lavage; BE, Bronchial Epithelium; BL, Bronchial Lumen; CaCl2, Calcium Chloride; cAMP, Cyclic Adenosin monophosphate; CB2, Cannabinoid receptor 2; CPCSEA, Committee for the Purpose of Control and Supervision Of Experiments on Animals; DPPH, 1, 1-diphenyl-2picrylhydrazyl; E, Edema; EC50, Effective concentration 50%; EDTA, Ethylene diamine tetraacetic acid; EGTA, Ethylene glycol-bis(b-aminoethyl ether) N,N,N0 ,N0 -tetraacetic acid; EOPI, Essential Oil of Pistacia integerrima J.L. Stewart ex. Brandis; FAD, Flavin Adenine Dinucleotide; FP, Fibrous connective tissue Proliferation; g, Gram; GC-MS, Gas chromatography and Mass Spectroscopy; H2O2, Hydrogen peroxide; HB, Hyperplasia of Bronchial Associated Lymphoid Tissues; i.p., Intraperitoneal; IC50, Inhibitory concentration 50%; KCL, Potassium Chloride; Kg, Kilogram; L, Liter; LD50, Lethal Dose 50%; LDH, Lactate dehydrogenase; LI, Alveolar luminal infiltration; L-NAME, L-NGNitroarginineMethyl Ester; LOX, Lipoxygenase; LPS, Lipopolysaccharide; LT, Leukotriene; M, Molar; mg, Milligram; MgCl2, Magnesium chloride; Ml, Milliliter; mM, Milimolar; mM, Milimole; MPO, Myleoperoxidase; Na þ , Sodium; NaCl, Sodium Chloride; NADPH, Nicotinamide adenine dinucleotide phosphate; NaH2PO4, Sodium Dihydrogen Phosphate; NaHCO3, Sodium bicarbonate; NaNO2, Sodium nitrate; NaOH, Sodium hydroxide; NF-ĸβ, Nuclear factor ĸβ; ng, nanogram; nm, Nanometer; NO, Nitric oxide; NOs, Nitric oxide synthase; OECD, Organisation for Economic Co-operation and Development; OVA, Ovalbumin; p.o., Peroral; PAF, Platelet activating factor; PBS, Phosphate Buffer Saline; PDE IV, Phosphodiesterase IV; PG, Prostaglandin; PI, Peribronchiolar infiltration; PSGL, P- selectin glycoprotein ligand; RANTES, Regulated on Activation, Normal T Expressed and Secreted; RNA, Ribose nucleic acid; rpm, revolution per minute; s, Second; SEM, Standard Error of Mean; SI, Interalveolar Septal Infiltration; TNF-α, Tumor necrosis factor α; UV, Ultravoilet; V, Blood Vessel; v/v, Volume by volume; VCAM, Vascular cell adhesion molecule; VEGF, Vascular endothelial growth factor; w/v, Weight by volume n Corresponding author. Tel.: þ 91 022 2667 0871/26671027; fax: þ91 022 2667 0816. http://dx.doi.org/10.1016/j.jep.2014.02.009 0378-8741 & 2014 Published by Elsevier Ireland Ltd.

Please cite this article as: Shirole, R.L., et al., Investigation into the mechanism of action of essential oil of Pistacia integerrima for its antiasthmatic activity. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.02.009i

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1. Introduction Asthma is a chronic inflammatory disease characterized by airway hyper responsiveness and recurrent reversible airway obstruction, affecting millions of people worldwide (WHO, 2002; Wong and Koh, 2000). The asthmatic reactions are the consequence of the release of granular mediators (e.g. histamine and 5-hydroxytryptamine), newly synthesized mediators (e.g. leukotrienes prostaglandins and platelet activating factor), and cytokines such as interleukins and tumor necrosis factor. The enzyme, phosphodiesterase IV (PDE IV) is predominant in inflammatory cells, including mast cells, eosinophils, macrophages, T lymphocytes and structural cells, which degrades cyclic nucleotides that are important messengers of cellular signaling and also plays a major role in homeostasis (Campos et al., 2003). Contemporary treatment of this disease addresses the dual need for rapid symptom relief (reliever therapy) while also attacking the inflammatory component of asthma (controller therapy) (Fernandes et al., 1999). Given the complexity of the disease process involved in asthma and activities of mediators linked to the disease, the case for a single mediator approach to asthma therapy is difficult to establish (Adamko et al., 2003). Medicinal plants have been used for various ailments by an alternative system of medicine and folklore treatments. However, many ethnopharmacological uses are yet to be justified scientifically for the rational and safe use. Further, the phytoconstituents present in the plants are one of the sources for new chemical entities in new drug discovery (Kumar et al., 2007). Pistacia integerrima J.L. Stewart ex. Brandis (Anacardiaceae) galls have been valued in traditional medicine in India for the treatment of asthma, chronic bronchitis, phthisis and other ailments for the respiratory tract, diarrhea, hiccough, vomiting of children, skin diseases, psoriasis, fever, to increase appetite and to remove bed humors (Chopra et al., 1958). It has also been reported in Ayurvedic compendium as follows.

and kaempferol-3-O-(4”-O-galloyl)-α-L-arabinopyranoside have been reported in leaves (Ahmad et al., 2008). Essential oil from Pistacia integerrima Stewart ex Brandis galls grown in India has not been explored yet for its biological effects related to medicinal use in hyperactivity of gut and airways disorders. From these reported activities and chemical constituents it can be seen that it may have an effect on inflammatory conditions of bronchial asthma. Therefore, in the present study, antiasthmatic activity of essential oil from galls has been evaluated and the possible mechanism of action is identified.

2. Materials and methods 2.1. Drugs and standards The following reference chemicals were obtained from the sources specified: ethylene glycol-bis (b-aminoethyl ether) N,N, N0 ,N0 -tetraacetic acid, o-dianisidine, nitrate reductase, naphthylethylenediamide, ovalbumin (Grade V), aluminum hydroxide gel, roflumilast, compound 48/80 and DPPH were purchased from Sigma-Aldrich Co., USA. LDH, FAD and NADPH were purchased from SRL Diagnostics Ltd., India, Heparin (Biological Evans, India). S(-)-Bay K 8644 and ketotifen fumarate were gift samples received from Bayer Healthcare AG, Germany and Sun Pharmaceuticals Industries Ltd., India, respectively. Other reagents were of reagent grade and were purchased from Sd fine-Chem Limited, India. 2.2. Procurement and extraction of essential oil of Pistacia integerrima J.L. Stewart ex Brandis Pistacia integerrima J.L. Stewart ex Brandis dried galls were collected from local distributor of Mumbai, India in April 2010 and were authenticated by Dr. Ganesh Iyer. A voucher specimen (no. 007) was deposited in the herbarium Ramnarain Ruia College, Bombay University, India. Essential oil of Pistacia integerrima J.L. Stewart ex Brandis galls (EOPI) was obtained from 500 g dried and powdered galls by hydro distillation as described by using Clevenger's apparatus (Clevenger, 1928). 2.3. Characterization of Pistacia integerrima J.L. Stewart ex Brandis galls

It balances Kapha and Vata (Kaphavatahara) and is useful in chronic respiratory disorders (Kshayahara) and fever (Jwarahara). It is used in those diseases wherein Vata in the chest region is misdirected towards upwards, such as Shwasa (asthma, bronchitis), Kasa (cough), Hikka (hiccup), and Vamana (vomiting). It is also useful in Aruchi (anorexia), and Trut (excessive thirst). It is also used in the treatment of Atisara (diarrhea) and Asrapitta (bleeding disorders) (Mishra, 1969). The essential oil has been reported to exhibit CNS depressant (Ansari et al., 1993a), antispasmodic, carminative and antibacterial, antiprotozoal, antiamoebic and anthelmintic activities (Khare, 2004), analgesic, anti-inflammatory activities (Ansari et al., 1994a; Ansari and Ali, 1996; Ahmad et al., 2010a) and hyperuricemia effect (Ahmad et al., 2008). The phytochemical investigation of Pistacia integerrima J.L. Stewart ex Brandis showed the presence of pistaciaphenyl ether and pisticiphloro-glucinyl ether in galls (Ahmad et al., 2011). Leaves contain carotenoids, triterpenoids and catechins (Ansari et al., 1993b, 1994b), n-decan-30 -ol-yl-n-eicosanoate, n-octadecan-9,11diol-7-one and 3-oxo-9β-lanost-1,20(22)-dien-26-oic acid and βsitosterol (Ahmad et al., 2010b). Polyphenolic compounds such as rutin, quercetin-3-O-β-D-glucopyranoside, kaempferol-3-O-β-D-glucopyranoside, quercetin-3-O-(6”-O-syringyl)-β-D-glucopyranoside

2.3.1. Specific gravity and refractive index of EOPI Specific gravity of essential oil of Pistacia integerrima J.L. Stewart ex Brandis was measured using pycnometer. Refractive index of 10 mg/ml EOPI solution in methanol was measured using a refractometer. 2.3.2. Determination of chemical composition of EOPI GC/MS analysis was carried out on a Hewlett-Packard 5970A mass selective detector, directly coupled to a HP 5790A gas chromatograph. A 26 m  0.22 mm column, coated with 0.13 mm of CP-Sil 5CB was employed, using helium carrier gas. The oven temperature was set to increase gradually from 60 1C (maintained for 3 min) to 250 1C at the rate of 5 1C/min and highest temperature 250 1C was maintained for 30 min at the end of the cycle. Electron Ionization (EI) mass spectra were acquired over a mass range 10–400 Da at 2 /s rate. Mass spectral survey was performed using Mass Spectral Library (NIST, 1998). 2.3.3. Animals Female Sprague-Dawley rats (175715 g), male Dunkin Hartley guinea pigs (300–350 g) and Swiss albino mice (18–20 g) were purchased from Haffkine's Institute, Mumbai, India. All animals were housed in standard polypropylene cages with wire mesh top

Please cite this article as: Shirole, R.L., et al., Investigation into the mechanism of action of essential oil of Pistacia integerrima for its antiasthmatic activity. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.02.009i

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and husk bedding and maintained under standard conditions of temperature (23 72 1C) and relative humidity (60 75%) with 12 h light: dark cycle. Animals were fed with commercially available standard rodent pellet diet. Water was provided ad libitum to the animals. Animals were acclimatized to laboratory conditions before the tests. All experiments were carried out between 09:00 and 17:00 h. For all animal experimentation protocols (Protocol nos. 23 and 24/2009) prior approvals were obtained from ‘Institutional Animal Ethics Committee’, of Bombay College of Pharmacy, Mumbai (registration number CPCSEA-BCP/2010/21) and all studies were performed in accordance with ‘Committee for the Purpose of Control and Supervision on Experiments on Animals’ (CPCSEA) guideline, Government of India on animal experimentation.

fumarate 0.1 ml of 10 μg/ml; final concentration 0.33 mg/ml [used as standard antianaphylactic, antihistaminic, inhibition of compound 48/80 induced Ca þ þ uptake in mast cells (Zaditor™, 1999 NDA 21- 066)]. All treatments were incubated for 15 min at 37 1C. Compound 48/80 (0.1 ml solution from 10 μg/ml, final concentration 3.33 mg/ml) was added to each group except Group 1. After further incubation for 10 min at 37 1C, the cells were stained with 0.1% w/v toluidine blue solution made in saline and examined using a Motic microscope (Motic SD digital microscope, Hong Kong). Percent protection of the mast cells in control group and treated groups were calculated by counting number of degranulated mast cells from total of at least 100 mast cells counted (Nair et al., 1995).

2.3.4. Eggs White Leghorn breed fertilized eggs (50–60 g) were purchased from local poultry farm, India.

2.7. In vitro evaluations on guinea pig ileum

2.4. Free radical (DPPH) scavenging activity For free radical (DPPH) scavenging activity as described by Blois (1958) briefly, 1 ml of EOPI in methanol (10–100 μg/ml) was added to 1 ml of DPPH 0.1 mM solution and incubated in dark for 20 min. Each value represents as mean 7SEM of five determinations. The absorbance was recorded at 517 nm using a UV spectrophotometer (Jasco, V-530, Japan). 2.5. Effect of EOPI on lipoxygenase activity The effect of EOPI on lipoxydase enzyme activity was determined by an in vitro method using soyabean lipoxydase as the enzyme and linoleic acid as the substrate as described by Shinde et al. (1999). In brief, the substrate solution consists of 0.05 ml linoleic acid emulsified with 0.05 ml ethanol and water up to 50 ml water. For the enzyme assay, 5 ml of this solution was diluted with 30 ml of 2 M borate buffer, pH 9. EOPI (0.05 ml), to obtain final concentrations range 5–30 μg/ml, were preincubated with the enzyme (0.05 ml; a solution of approximately 10,000 units/ml of lipoxydase in ice cold 2 M borate buffer, pH 9) for 5 min. Lipoxygenation was started by addition of the 0.05 ml enzyme solution to the 2 ml substrate solution and change in absorbance was determined per minute at 234 nm using a spectrophotometer for a time period of 5 min. Six determinations were performed for each concentration. Phenidone (1–30 ng/ml) was used as a reference standard for a comparison. The percent inhibition was calculated from the equation.

Guinea pigs were fasted for 18 h with water ad libitum before sacrifice by carbon dioxide asphyxia. 2 cm pieces were dissected from the ileum segment 10–20 cm near the ileocecal junction, mounted for tension recording and allowed to equilibrate for 1– 2 h in 20 ml chambers containing Tyrode solution (composition in mM: NaCl 136.0, KCl 5.0, MgCl2 0.98, CaCl2 2.0, NaH2PO4 0.36, NaHCO3 11.9 and glucose 5.5), pH 7.4, maintained at 37 1C, and bubbled with carbogen. In experiments with calcium-free medium, CaCl2 was omitted from the normal Tyrode solution and 0.2 mM ethylene glycol-bis (b-aminoethyl ether) N,N,N0 ,N0 -tetraacetic acid (EGTA) was added. The mechanical responses in ileum were isometrically recorded with a force transducer (MLT 050/D) PowerLab Data acquisition system (AD Instruments, Australia) using Chart 5 software. The contractile amplitude was measured at the peak deflection.

2.7.1. Effect of EOPI on histamine, acetylcholine and KCl induced contraction of isolated guinea pig ileum To determine the effect of EOPI on histamine and ACh induced contraction of isolated guinea pig ileum (Ghosh, 2005; Magalhãeset al., 2004) response to 60 mM K þ was recorded as maximum amplitude of contraction of tissue. Sub-maximal dose of histamine and ACh (approximately 70% of maximum contraction induced by 60 mM K þ ) was selected for further studies. The ileal tissues were preincubated with EOPI (10–300 μg/ml) for 10 min and response to histamine (0.2–0.6 μM) and ACh (0.1–0.5 μM) was recorded. Response of histamine and ACh was expressed as percentage of 60 mM K þ induced contraction. IC50 values were calculated using GraphPad Prism 5 (GraphPad Software Inc.).

% inhibition ¼ ðAcontrol –Atest Þ=Acontrol  100 2.6. Effect of EOPI on compound 48/80-induced histamine release from mast cells In four female rats (175 715 g) 20 ml normal saline containing 5 unit/ml of heparin was injected in the peritoneal cavity under light ether anesthesia. After a gentle abdominal massage, the peritoneal fluid containing mast cells was collected in centrifuge tubes placed over ice. Peritoneal fluid of all rats were pooled and centrifuged at 2000 rpm for 5 min. Supernatant solution was discarded. The cells were washed twice with saline and resuspended in 1 ml of saline. Peritoneal cell suspension (0.1 ml, 1  105 cells/mm3) was transferred to 6 groups and treated as follows. Group 1 – Saline control (0.2 ml), Group 2 – Compound 48/ 80 þ0.1 ml Saline, and Groups 3–5 – EOPI 0.1 ml of 10, 30 and 100 mg/ml EOPI solution to achieve final concentration of 3.33 mg/ ml, 10 mg/ml and 33.33 mg/ml, respectively. Group 6 – ketotifen

2.7.2. Effect of EOPI on ACh-induced contraction of the isolated Guinea pig ileum under calcium-free conditions For assessment of effect of EOPI on ACh-induced contraction of the isolated guinea pig ileum under calcium-free conditions (Magalhães et al., 2004), the ileal tissue was contracted with 60 mM K þ and the maximum amplitude of contraction was recorded. Organ bath was filled with Ca2 þ free Tyrode solution and response for ACh (60 mM) induced contraction was recorded after 5 min. The contractile response of ACh was expressed as percent contraction of 60 mM K þ in normal Tyrode solution. The tissue was rinsed with Ca2 þ free Tyrode solution and further response for ACh (60 mM) was recorded. The above protocol was repeated so as to get three consecutive responses of ACh (60 mM) in Ca2 þ free Tyrode solution. EOPI (80, 160 and 320 mg/ml) was incubated for 10 min in Ca2 þ free Tyrode solution and response of ACh (60 mM) was recorded.

Please cite this article as: Shirole, R.L., et al., Investigation into the mechanism of action of essential oil of Pistacia integerrima for its antiasthmatic activity. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.02.009i

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2.7.3. Effect of EOPI on S(-)-Bay K 8644-induced tonic contractions To study the effect of EOPI on S(-)-Bay K 8644-induced tonic contractions (Conte Camerino et al., 1987), the ileum was partly depolarized by addition of 15 mM KCl for 10 min. In the presence of KCl, a contraction with S (-)-Bay K 8644 (10  6 M), a selective Ltype Ca2 þ channel agonist, was induced. During the stabilization of tonic phase of this contraction, EOPI (32–256 mg/ml) was added cumulatively in order to obtain a concentration response curve. 2.8. Angiogenesis Effect on EOPI on angiogenesis was studied as described previously (Krenn, 2009). In brief, fertilized white Leghorn eggs were incubated at 37 1C with 80% humidity. The eggs were divided into seven groups of six eggs each and received the following treatment: Group I: Saline control; Group II: Vehicle control (Tween 80, 0.01% v/v); Group III: Positive control (Erythropoietin (100 μg/ml)); Group IV: Heparin (100 U/ml) þ Erythropoietin (100 μg/ml); Group V: EOPI 25 μg/ml þErythropoietin (100 μg/ ml); Group VI: EOPI 50 μg/ml þ Erythropoietin (100 μg/ml); and Group VII: EOPI 100 μg/ml þErythropoietin (100 μg/ml). On day 6, these eggs were inoculated with 25–100 μg/ml of EOPI through a window made in the eggshell in upward position near the air sac and sealed with parafilm. The eggs were then incubated under same conditions in the incubator till day 12. On day 12 the eggs were broken from the region of air sac and window made for inoculation. The CAM was isolated and then spread out in the petriplate containing saline and images of four distant areas were taken with the help of the stereomicroscope. The number of blood vessels and the bifurcations in each area in each image were counted using the image analysis software. An average of number of blood vessels of all the four images of the same CAM was calculated. The number of blood vessels was treated to be directly proportional to an extent of angiogenesis. 2.9. Acute oral toxicity of EOPI Acute toxicity studies were performed as per OECD (2001) guideline 425 on Swiss albino mice. Briefly, the 4 h fasted Swiss albino mice were administered EOPI (dose progression: 2000 (n ¼3); 300 and 175 mg/kg, i.p. n ¼ 6) observed for mortality. The animal behavior and mortality index were closely observed for first 3 h then at an interval of 4 h during next 48 h. LD50 was determined using AOT425 statpgm (Version: 1.0). 2.10. Effect of EOPI on lipopolysccharide (LPS) induced lungs inflammation in rats Effect of EOPI on LPS induced lungs inflammation in rats was as described previously (Spond et al., 2001). In brief, thirty six Sprague-Dawley female rats were divided randomly in six groups of six animals each. EOPI (7.5, 15 and 30 mg/kg) was administered intraperioneally for four days to animals prior to LPS administration (The doses were selected based on LD50 value and preliminary efficacy studies). Roflumilast (1 mg/kg, p.o. for 4 days), a PDE4 inhibitor was used as standard drug for comparison. Solution of LPS (Escherichia coli, O111:B4; 1 mg/ml) was prepared in sterile phosphate buffered saline (PBS), pH 7.2 just prior to administration to animals. LPS solution was maintained cold by keeping in ice throughout the administration procedure. Animals were anesthetized with ketamine: xylazine (80:20 mg/kg, i.p.) and administered intratracheally with LPS or sterile PBS depending on the group to which they belong. Animals were allowed to recover on a heat pad, returned to housing and permitted access to food and water ad libitium. Four hours after the provocation, terminal anesthesia was induced with urethane (1.2 g/kg, i.p.), trachea

was cannulated and bronchoalveolar lavage was performed with 2  2 ml of ice cold PBS. Bronchoalveolar lavage (BAL) fluid was kept in ice immediately after the collection. Lung biopsies were taken after BAL fluid collection. BAL fluid was assessed for the total cell count, neutrophil count, nitrite/nitrate, total protein and albumin. Myeloperoxidase content in the lungs homogenate was estimated. 2.10.1. Cell count BAL fluid was vortexed and aliquote of 100 μl was taken for total cell counts using equal volume of Turk's solution. 2.10.2. Neutrophil count The cell pallet after centrifugation was suspended in rat serum (harvested previously) and smears were prepared. Cells were stained with May-Grünwald Giemsa stain and neutrophil 100 cells were counted using standard morphological criteria. 2.10.3. Measurement of myeloperoxidase activity For myeloperoxidase estimation, as described by Bradley et al. (1982) briefly, pieces of lung were excised, rinsed with ice cold saline, blotted dry and weighed. Minced tissue was homogenized in 10 volumes of ice-cold potassium phosphate buffer (pH 7.4), using a Remi tissue homogenizer (RQ-127A). The homogenate was centrifuged at 3500 rpm for 30 min at 4 1C (Remi centrifuge C23). The supernatant was discarded. 10 ml of ice-cold 50 mM potassium phosphate buffer (pH 6.0), containing 0.5% w/v hexadecyltrimethyl ammonium bromide (HETAB) and 10 mM EDTA were added to the pellet. It was then subjected to one cycle of freezing and thawing and brief period (15 s) of sonication. After sonication, the solution was centrifuged at 15,000 rpm for 20 min (Remi centrifuge, C23). Myeloperoxidase activity was measured spectrophotometrically as follows: 0.1 ml of supernatant was mixed with 2.9 ml of 50 mM phosphate buffer containing 0.167 mg/ml Odianisidine hydrochloride and 0.0005% v/v H2O2. The change in absorbance was measured spectrophotometrically (Shimadzu UV 160A UV–vis spectrophotometer), at 460 nm. One unit of MPO activity was defined as the change in absorbance per minute by 1.0 at room temperature, in the final reaction mixture. The MPO activity was calculated using the following equation: MPO activity ðU=gÞ ¼ X= wt of the piece of tissue taken where X ¼10  change in absorbance per minute /volume of supernatant taken in the final reaction. 2.10.4. Measurement of nitrite/nitrate Nitrite/nitrate (NOx) production, an indicator of NO synthesis, was measured in BAL fluid as described by Leza et al. (1998). Briefly, to reduce nitrate to nitrite, nitrate in BAL fluid supernatant was incubated with nitrate reductase (0.1 U/ml) and NADPH (1 mM) and FAD (50 μM) at 37 1C for 15 min followed by another incubation with LDH (100 U/ml) and sodium pyruvate (10 mM) at 37 1C for 5 min. Nitrite in the samples was measured by the Griess reaction, by adding 100 μl of Griess reagent (0.1% w/v naphthyl ethylenediamine dihydrochloride in water and 1% w/v sulfanilamide in 5% w/v concentrated H2PO4; mixed in volumes 1:1) to 100 μl samples. The optical density at 550 nm was measured using a microplate reader (Modulus Microplate Reader, Turner Biosystem, USA). Nitrate concentrations were calculated by comparison with the absorbance of standard solution of sodium nitrate prepared in saline solution. 2.10.5. Total protein assay As a measure of epithelial injury and lung permeability, total protein concentration in BAL fluid was measured by the Lowry

Please cite this article as: Shirole, R.L., et al., Investigation into the mechanism of action of essential oil of Pistacia integerrima for its antiasthmatic activity. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.02.009i

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method (Lowry et al., 1951). In brief, 100 ml BAL fluid supernatant was mixed with 2 ml Lowery reagent (Reagent A: 0.1 N NaOH, 2 g Sodium carbonate and 20 mg sodium potassium tartarate in 100 ml distilled water Reagent B: CuSO4 1 g in 100 ml distilled water. Mixed Reagent A 100 ml and B 2.5 ml) and incubated for 10 min at room temperature. To this mixture 0.2 ml of Folin's reagent (1:2 dilution of commercially available Folin's Reagent) was added and incubated at 30 1C. The optical density at 660 nm was measured using a microplate reader (Modulus Microplate Reader, Turner Biosystem, USA) and total protein was quantified using a standard plot of bovine serum albumin (0.1–1 mg/ml). 2.10.6. Albumin assay Albumin measurements in BAL fluid were performed with a bromocresol green method (Doumas and Biggs, 1972). Briefly, aliquots of BAL fluid supernatant (50 ml) were incubated for 10 min at room temperature with 500 ml of albumin working reagent (bromocresol green 0.08 mmol/L, succinate buffer pH 4.2 70.1 at 25 1C and sodium azide 1 g/L). Absorbance was read at 630 nm (Modulus Microplate Reader, Turner Biosystem, USA) and albumin was quantified using a standard plot of bovine serum albumin (0.1 to 1 mg/ml). 2.10.7. Histopathological studies The lower lobe of the right lungs was dissected and fixed in 10% v/v buffered formalin. The specimens were subjected to sectioning, staining, mounting and observation. Briefly, after a week tissues were washed thoroughly in repeated changes of 70% alcohol and then dehydrated in ascending grades of alcohol (70 to 100% v/v). After dehydration, the tissues were cleaned in xylene and embedded in paraffin wax. Processed tissues were sectioned (5 μm) and taken on clean glass slides and stained by hematoxylin and eosin dyes. Stained slides were examined under a Olympus BX10 optical microscope attracted with Olympus DP12 camera (Olympus, Japan).

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w/v for 10 s). The respiratory flow due to gasping was monitored over a period of next nine minutes for all the treated guinea pigs and compared with ovalbumin control group.

3. Statistical analysis Results were expressed as mean 7SEM. The inter-group variation was measured by Student's t-test between two groups and one-way analysis of variance (ANOVA) followed by Dunnett's test for more than two treatments. Statistical significance was considered at p o0.05. The concentration of the agent producing 50% of the maximal contraction (EC50) or inhibition (IC50) in individual experiment was determined graphically from a concentration response curve for contraction or relaxation response, respectively. Statistics was applied using GraphPad Prism 5 software version 5.00 (GraphPad Software Inc.).

4. Results 4.1. Characterization of EOPI The EOPI was found to be colorless liquid with terebinthine odor and strongly astringent taste. The yield of extraction of EOPI was found to be 1.3%. Specific gravity of EOPI was found to be 0.889, while refractive index was found to be 1.215. GCMS chromatograph of EOPI revealed chemical composition as mentioned in Table 1. 4.2. Free radical scavenging activity EOPI showed concentration dependant free radical (DPPH) scavenging activity up to 100 mg/ml. Maximum inhibition of 44.937 2.53% was observed at 100 mg/ml concentration. 4.3. Effect of EOPI on lipoxygenase activity

2.11. Effect of EOPI on ovalbumin induced bronchoconstriction in guinea pigs To investigate the effect of EOPI on ovalbumin induced bronchoconstriction, 30 male guinea pigs (300–350 g) were actively sensitized with ovalbumin (OVA) 1 mg/ml adsorbed over 20% aluminum hydroxide gel in sterile saline. Each guinea pig received 0.125 ml of above suspension in each paw (sub-plantar region) and 0.5 ml subcutaneously (Hutson et al., 1988). Twenty one days after sensitization, each guinea pig was placed in a Whole Body Plethysmograph (WBP) Chamber and habituated with aerosol of saline for 5 min. The aerosol was generated by a nebulizer connected to the chamber which delivered particles of 0.5– 10 μM diameter at rate of 0.4 ml/min. Fresh air was supplied constantly into the chamber at the rate of 0.15 L/s throughout the experiment. On 22nd day, each animal was challenged with 0.01% w/v ovalbumin aerosol for 10 s, which produced bronchoprovocative convulsion and recovered over a period of time as evidenced by the normal breathing pattern. The respiratory flow due to gasping was monitored over a period of next 9 min for each animal. The animals which showed preconvulsion by 2–4 min were selected for the study. Thirty guinea pigs were divided into five groups of five animals each, viz. Group 1 ovalbumin control, Groups 2–4 EOPI, 7.5, 15 and 30 mg/kg, i.p. for four days respectively, Group 5 roflumilast 1.3 mg/kg, p.o. for four days. Seven days after the above challenge, the guinea pigs were exposed to the test drugs and roflumilast. On fourth day one hour post-treatment guinea pigs were transferred to a WBP chamber and challenged with ovalbumin aerosol (0.01%

Lipoxygenase produced peak enzyme activity at 3rd minute after addition of substrate, and linolic acid. Phenidone (1–30 ng/ ml) and EOPI (5–30 mg/ml) dose dependently inhibited the rate of lipoxygenase kinetics and peak enzyme activity with IC50 of 7.81 ng/ml and 19.72 mg/ml respectively. 4.4. Compound 48/80-induced mast cell degranulation Compound 48/80 (0.1 ml solution from 10 μg/ml) produced 78.21 71.4% disruption of mast cells compared to saline control. Pre-treatment with EOPI (3.33, 10 and 33.33 mg/ml) significantly Table 1 GCMS chromatograph of EOPI. Retention time

Area (%)

Name of compound

4.42 4.55 4.96 6.44 6.52 6.78 6.96 7.21 7.27 7.88 8.05 8.35 8.41 10.00

00.73 01.37 11.54 10.27 17.06 11.93 08.90 02.75 13.99 03.88 05.33 00.77 01.61 06.35

m-Cumenol α-Terpinene Cymene Tetrahydrocarvone 4-Carvomenthenol L-terpinen-4-ol Borneol α-Terpinene Levo-bornyl acetate β-caryophyllene β-caryophyllene Cuminaldehyde Hexanoic acid (-)-Spathulenol

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(p o0.05) reduced mast cell degranulation to 47.3373.32%, 32.1 72.01% and 19.08 70.47% respectively. Ketotifen (0.33 mg/ ml) significantly (p o0.05) reduced mast cell degranulation to 14.10 70.34%. Ketotifen (0.33 mg/ml) exhibited 81.97% protection of compound 48/80 induced mast cell degranulation.

4.5. In vitro evaluations of EOPI on isolated guinea pig ileum 4.5.1. Effects of EOPI on the contractions induced by ACh, histamine, and KCl in isolated guinea pig ileum Pre-incubation of ileal preparation with EOPI (10–300 mg/ml) for 10 min resulted in decreased amplitude of contractions induced with 60 mM K þ , sub-maximal (inducing responses approximately 70% of the maximum) concentrations of ACh (0.1– 0.5 mM) and histamine (0.2–0.6 mM). EOPI inhibited the contractions at all tested concentrations with the IC50 values of 79.26 mg/ ml for K þ , 106.0 mg/ml for ACh and 220.60 mg/ml for histamine (Fig. 1).

4.5.2. Effect of EOPI on ACh-induced contraction of the isolated guinea pig ileum under calcium-free conditions The amplitude of the contraction induced by ACh (60 mM) in Ca2 þ free Tyrode solution (with 0.2 mM EGTA) suggests Ca2 þ release from intracellular stores. The contraction induced at 5 min after Ca2 þ removal was 31.80 71.67% of the control response to 60 mM K þ under normal conditions (Ca2 þ containing Tyrode solution). Pre-incubation of EOPI 80, 160 and 320 mg/ml further significantly (p o0.05) reduced the amplitude of contraction in Ca2 þ -free Tyrode solution to 13.72 71.26%, 7.11 70.59% and 3.097 0.48% of K þ induced contraction, respectively. The effect was found to be reversible as ACh contractile response was reproduced after washing out EOPI. 4.5.3. Effect of EOPI on S(-)-Bay K 8644-induced tonic contractions Cumulative addition of EOPI (32–256 mg/ml) on tonic component of the contractions elicited by S-(-)-Bay K 8644 (10  6 M) resulted in a concentration dependant relaxation (EC50 ¼ 100 mg/ ml) of isolated guinea pig ileum (Fig. 2). 4.6. Effect of EOPI on angiogenesis Erythropoietin group showed significant increase in number of blood vessels formation compared to vehicle control. The drug treated and heparin treated group showed significant (p o0.05) reduction in number of blood vessels formation. The angiostatic activity of the drug treated group was found to be comparable with the heparin treated group (Fig. 3). 4.7. Acute toxicity of EOPI

Fig. 1. Concentration-dependent inhibitory effects of EOPI on submaximal contractions induced by acetylcholine (0.1–0.5 mM), histamine (0.2–0.6 mM) and K þ Q9 (60 mM); each values represents mean7 SEM of six determinations.

Fig. 2. Effect of EOPI on the tonic contraction elicitesd by 15 mM KCL and S-(-)-Bay K 8644. Each point represents mean 7SEM of six determinations.

The estimated LD50 of EOPI was found to be 229.1 mg/kg i.p. in Swiss albino mice. The animals showed depression at lower doses.

Fig. 4. Effect of EOPI and Roflumilast on total cell count. Each value represents mean7 SEM of six rats. Statistical comparison was performed by one way ANOVA followed by Dunnett's test, # po 0.05 different from saline control group; n po 0.05 different from LPS control group.

Fig. 3. Effect of EOPI on angiogenesis. Each value represents mean 7 SEM of six determinations. Statistical comparison was performed by one way ANOVA followed by Dunnett's test, n p o0.05 different from erythropoietin group.

Please cite this article as: Shirole, R.L., et al., Investigation into the mechanism of action of essential oil of Pistacia integerrima for its antiasthmatic activity. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.02.009i

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At higher doses agitation, clonic convulsions and respiratory distress preceded death were observed. 4.8. LPS induced lungs inflammation in rats 4.8.1. Effect of EOPI on total cell count in BAL fluid Intratracheal instillation of LPS significantly (p o0.05) increased total cell count in BAL fluid in comparison to saline control. The drug treated and roflumilast treated groups showed significant (p o0.05) reduction in total cell count in BAL fluid (Fig. 4). 4.8.2. Effect of EOPI on neutrophils count in BAL fluid Further investigation of BAL fluid revealed significant (p o0.05) increase in number of neutrophils from saline control 17.27 72.54% to 75.6972.67% four hour after intratracheal instillation of LPS. Pre-treatment with EOPI 7.5, 15 and 30 mg/kg, i.p. and roflumilast 1 mg/kg, p.o. significantly (p o0.05) reduced neutrophil count to 53 72.66%, 41 72.20, 37 71.64% and 22 73.30% respectively in comparison to LPS control.

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4.8.3. Effect of EOPI on MPO activity Intratracheal instillation of LPS significantly (p o0.05) increased MPO activity from 24.16 78.79 units/g observed in saline control to 228.667 16.74 units/g. Pre-treatment with EOPI 7.5, 15, 30 mg/kg, i.p. and roflumilast significantly (p o0.05) reduced the MPO levels in the BAL fluid to 139.00 724.52, 121.28 730.38, 113.41 710.95 and 128.44 722.95 units/g, respectively in comparison to LPS control. 4.8.4. Effect of EOPI on nitrate/nitrite content in BAL fluid The nitrate/nitrite assay showed significant (po0.05) increase in nitrate/nitrite in LPS control group compared to saline control. The drug treated and roflumilast showed significant decrease in nitrate/nitrite content in nitrate/nitrite in compared to LPS control group. Nitrate/nitrite concentration in EOPI 30 mg/kg, i.p. group was found to be similar to that of in roflumilast treated group (Fig. 5). 4.8.5. Effect of EOPI on total protein in BAL fluid The protein concentration in BAL fluid of LPS control was significantly (po 0.05) increased from 0.26 70.14 mg/ml in saline control to 1.85 70.29 mg/ml. Pre-treatment with EOPI 7.5, 15, 30 mg/kg, i.p. and roflumilast 1 mg/kg, p.o., showed significant reduction in protein concentration to 1.26 70.41, 1.06 70.48, 1.046 70.37 and 0.98 70.21 mg/ml, respectively in comparison to LPS control. 4.8.6. Effect of EOPI on albumin content in BAL fluid Intratracheal instillation of LPS significantly (po0.05) increased albumin concentration in BAL fluid from 0.1370.05 mg/ml of saline control to 1.1070.18 mg/ml. Pre-treatment with EOPI 7.5, 15, 30 mg/kg and roflumilast 1 mg/kg, p.o. significantly (po0.05) reduced the albumin levels to 0.7070.20, 0.6270.23 and 0.527 0.18 and 0.4670.19 mg/ml in comparison to LPS control.

Fig. 5. Effect of EOPI and Roflumilast on nitrate/nitrite levels in Bronchoalveolar Lavage fluid in LPS induced lung inflammation in rats. Each value represents mean 7 SEM of six rats. Statistical comparison was performed by one way ANOVA followed by Dunnett's test, # p o0.05 different from saline control group; n p o 0.05 different from LPS control group.

4.8.7. Histopathological evaluations Histological examination of saline control group showed no abnormality in the lungs. In LPS control group very severe peribronchiolar, interalveolarseptal and perivascular infiltration

Fig. 6. Histopathological Evaluation of lungs in LPS induced lung inflammation in rats. V ¼ Blood Vessel, FP ¼Fibrous connective tissue Proliferation, SI¼ Interalveolar Septal Infiltration, E ¼ Edema, PI¼ Peribronchiolar infiltration, LI¼ Alveolar luminal infiltration, AS ¼ Inter Alveolar Septum, A ¼Alveoli, BL ¼ Bronchial Lumen, BE ¼Bronchial Epithelium, HB¼Hyperplasia of Bronchial Associated Lymphoid Tissues.

Please cite this article as: Shirole, R.L., et al., Investigation into the mechanism of action of essential oil of Pistacia integerrima for its antiasthmatic activity. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.02.009i

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Fig. 7. Effect of EOPI on ovalbumin induced bronchoconstriction in guinea pigs. Each value represents mean 7SEM of five guinea pigs. Statistical comparison was performed by one way ANOVA followed by Dunnett's test, np o 0.05 different from ovalbumin control group.

of inflammatory cells (majority were neutrophils) and edema (þ þ þ þ ) was found. The inflammatory cells were present in bronchiolar and alveolar lumen. There was hyperplasia of bronchial associated lymphoid tissues (þ þ ). There was severe proliferation of fibrous connective tissue in LPS treated lungs. Pretreatment with EOPI (7.5 mg/kg, i.p.) exhibited diffused lesions involving peribronchiolaredema (þ þ), interalveolarseptal and perivascular, peribronchiolar infiltration of neutrophils (þ þ). In EOPI (15 mg/kg, i.p.) treated group mild diffused lesions involving focal interalveolarseptal and intraluminal (alveolar) infiltration of neutrophils (þ þ ) were observed. No abnormality was found in rats pre-treated with EOPI (30 mg/kg, i.p.) and roflumilast (1 mg/ kg, p.o.) (Fig. 6). 4.9. Effect of EOPI on ovalbumin induced bronchoconstriction in guinea pigs In OVA control group nebulized with saline for 5 min duration, ovalbumin (0.01% w/v for 10 s) exposure produced first sign of bronchoprovocative convulsions at 33.06 76.28 s which reached peak bronchoprovocative response during two to three minutes. Pre-treatment with EOPI (7.5, 15 and 30 mg/kg, i.p.) and roflumilast (1.3 mg/kg, p.o.) significantly (p o0.05) abolished the bronchoprovocative response in sensitized guinea pigs at 2 min (Fig. 7).

5. Discussion The present study for the first time demonstrated that essential oil of Pistacia integerrima J.L. Stewart ex Brandis (EOPI) was effective in animal model of acute and chronic inflammatory conditions in bronchial asthma due to presence of tetracyclic triterpenoids (Ansari and Ali, 1996). EOPI showed in vitro inhibition of DPPH oxidation, 5-lipoxygenase, peritoneal mast cell degranulation, angiogenic in CAM assay and L-type voltage gated Ca channel on isolated guinea pig ileum activity. In vivo studies of EOPI in rats showed amelioration of LPS-induced lung injury, including inhibition of albumin, protein level, MPO activity and inflammatory cell recruitment in BAL fluid. Inhibition of allergen induced airway hyper-responsiveness in guinea pigs may contribute to anti-asthmatic activity of Pistacia integerrima J.L. Stewart ex Brandis. Reactive oxygen species (ROS) produced in asthmatic airways activates eosinophils, neutrophils, monocytes, and macrophages to generate superoxides (O3 ). ROS also activate NF-kB, amplifying the inflammatory response (Barnes et al., 1998). EOPI was thus

evaluated in vitro for its antioxidant potential using DPPH scaven67 ging assay. EOPI exhibited significant antioxidant activity at 10– 68 100 mg/ml. Our finding correlated well with results reported by 69 Joshi and Mishra (2010). The polyphenolic compounds have been 70 reported to show antispasmodic, antiasthmatic and anti71 inflammatory activity (Gopalakrishnan et al., 1980). The phyto72 chemical investigation of Pistacia integerrima J.L. Stewart ex 73 Brandis leaves revealed carotenoids, triterpenoids and catechins 74 beside flavonoid glycosides (Ansari et al., 1994b). The antioxidant 75 potential could be assigned to its high polyphenolic contents. 76 In the present investigation antiallergic activity of essential oil 77 of Pistacia integerrima J.L. Stewart ex Brandis galls (EOPI) was 78 evaluated using in vitro mast cell degranulation studies. Com79 pound 48/80, a calcium channel ionophore, which can induce mast 80 cell secretion to a site on the cell membrane, is associated with an 81 influx of Ca2 þ into the cell (Cheong et al., 1998). EOPI (33.33 mg/ml) 82 83 showed 80.92% protection of compound 48/80 induced mast cell 84 degranulation. Recently, Adusumalli et al. (2013) reported anti85 asthmatic activity of aqueous extract of Pistacia integerrima Linn by 86 70.29% protection of actively sensitized mesenteric mast cells 87 membrane stabilizing in albino rats at maximum dose of 54 mg/ 88 kg, p.o. for 14 days. Mast cells and basophils play a critical role in 89 Type I hypersensitivity and allergic reactions when activated 90 through IgE by specific antigens followed by release of inflamma91 tory mediators such as histamine, leukotrienes, and various 92 cytokines/ chemokines (Uvnas, 1969; Crossland, 1980). These 93 mediators initiate rapid vascular permeability, leading to plasma 94 extravasations, tissue edema, bronchoconstriction, mucus over95 production and leukocyte recruitment (Galli and Lantz, 1998; Galli and Wershil, 1996). Ansari and Ali (1996) have reported analgesic Q5 96 97 and anti-inflammatory activity of tetracyclic triterpenoids isolated 98 from Pistacia integerrima J.L. Stewart ex Brandis galls. Plants 99 containing flavonoids have been reported to show antihistaminic 100 and mast cell degranulation properties (Havsteen, 1983; Pathak 101 et al., 1991). 102 Further studies were conducted on isolated tissue preparation 103 where spasmolytic activity of EOPI on histamine, acetylcholine and 104 potassium chloride was evaluated using guinea pig ileum prepara105 tion. Recently, Adusumalli et al. (2013) reported only antihistami106 nic activity of aqueous extract of Pistacia integerrima Linn in vitro 107 on isolated guinea pig tracheal chain preparation. In the present 108 investigation, EOPI demonstrated a potential spasmolytic activity 109 as suggested by inhibition of histamine and acetylcholine induced 110 contractions in guinea pig ileum. ACh-induced contraction in 111 smooth muscle is mediated by a release of intracellular Ca2 þ from 112 sacroplasmic reticulum and by Ca2 þ entry via voltage dependant 113 and independent mechanisms (Bolton, 1979; McConalogue and 114 Furness, 1994). The abundantly expressed voltage gated Ca2 þ 115 channels in the guinea pig ileum are the L-type (Cav-L) (Bolton, 116 1979; Tomita, 1981) and Cav1.2 (Catterall et al., 2005). To evaluate 117 if the Cav channel was involved in the response of EOPI, the effect 118 of EOPI on S-(-)-Bay 8644-precontracted ileum was investigated. 119 S-(-)-Bay 8644, an L-type Cav agonist, acts by directly binding to 120 the channel's α-subunit and not by depolarization (Spedding and 121 Paoletti, 1992). Under these conditions EOPI induced a concentra122 tion dependant relaxation (EC50 ¼ 100 mg/ml), suggesting that the 123 voltage gated L-subtype calcium channel is involved in EOPI's 124 spasmolytic activity. FDA had approved selective human 5125 lipoxygenase inhibitor, zileuton, for the treatment of asthma 126 (Israel et al., 1996). EOPI shows in vitro inhibition of soyabean 127 lipoxygenase. Middleton et al. (2000) reported inhibition of a 128 number of other enzymes like cyclo-oxygenase (COX) and lipoox129 ygenase by terpenoids and flavonoids like compounds. 130 Excessive angiogenesis or neovascularization is an important 131 factor to pathogenesis of many chronic inflammatory diseases 132 including asthma in highly industrialized western countries. Thus

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anti-angiogenic strategies are emerging as one approach for treatment and prevention of chronic diseases (Krenn, 2009). An increase in vascularity in the bronchial mucosa of asthmatics has been a well described aspect of asthmatic airways. Inhaled glucocorticoids decrease the airway vascularity attenuating the increased blood flow (Barnes and Adcock, 2003; Caramori and Adcock, 2003). The Chorioallantoic Membrane (CAM) is used to study the macromolecules with angiogenic and antiangiogenic activity. An antiangiogenic response occurs within 72–96 h after stimulation in the form of increased vessel density around the implant of angiogenesis inducer like erythropoietin. Conversely, when angiostatic compound is tested the vessels become less dense around the implant and eventually disappear (Ribatti et al., 2000). In the present study EOPI inhibited erythropoietin induced angiogenesis. Activation of protein kinase c (PKC) was reported to induce CAM angiogenesis between9 and 11 days (Tsopanoglou et al., 1993), whereas increased level of intracellular cyclic adenosine monophosphate substantially down regulated this PKCmediated angiogenic stimulus (Tsopanoglou et al., 1994). In vitro studies made a strong reservation for evaluation of EOPI for its anti-inflammatory and anti-allergic activities in vivo. In vivo evaluation of EOPI was carried out in LPS induced acute lung inflammation (neutrophilia). LPS triggers activation of mononuclear phagocytes through a receptor mediated process, leading to the release of a number of cytokines, including tumor necrosis factor-α (TNF-α) (Yang et al., 1998) which has been suggested to play a critical role in the initiation, maintenance and progression of airway inflammation in asthma (Ohkawara et al., 1992). The increased adherence of neutrophils to endothelial cell induced by TNF-α leads to a massive infiltration in the pulmonary space (Kips et al., 1992). In the present investigation intratracheal instillation of LPS increases epithelial and endothelial permeability, influx of protein and albumin, WBC migration, MPO activity and nitrate/ nitrite levels. EOPI ameliorated the LPS induced WBCs migration, MPO activity and excessive production of proinflammatory mediators suggesting protective role of EOPI in bronchial asthma. Lefort et al. (1998) reported development of acute lung injury after intraperitoneal injection of bacterial LPS animal models which can be measured by albumin extravasation or neutrophils myeloperoxidase activity in the lung parenchyma. In the present investigation in vivo intra-tracheal administration of LPS in SD female rats induced an invasion of the airway lumen with neutrophils and increased myeloperoxidase activity. Infiltrating neutrophils were also activated after the LPS challenge as evidenced by the increased levels of MPO activity. EOPI was found to attenuate LPS induced neutrophilia in rats. EOPI inhibited leukocyte infiltration as a measure of total cell count in BAL fluid. Significant decrease in albumin extravasation was seen on albumin and protein levels in BAL fluid of EOPI treated rats. Decrease in leukocyte infiltration was associated with significant reduction in neutrophil count and myeloperoxidase activity in BAL fluid. EOPI was also evaluated for the effect on nitric oxide (NO) levels in BAL fluid. In the present investigation NO levels in the BAL fluid of EOPI treated animals were found to be significantly lower than LPS control, demonstrating the inhibitory influence of EOPI on NO in LPS induced lungs inflammation in rats. Endogenous NO produced by inducible NO synthase (iNOS), is well known for its possible role in inducing airway diseases including asthma (Barnes and Belvisi, 1993). NO strongly promotes the chemotaxis of inflammatory cells in lungs and favors the development of Th2 response (Taylor-Robinson et al., 1993). This is good evidence that NO inhibitors suppress airway inflammation by suppressing the recruitment of inflammatory cells and mucus secretion in the lungs (Barnes et al., 1998). In LPS-activated inflammatory cells such as macrophages, NF-ĸβ is known to be an important regulator of iNOS, TNFα and IL-1β expression (Xie et al., 1994). Recently, Mehla et al. (2011) found that

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ethyl gallate isolated from ethyl acetate fraction of Pistacia integerrima Linn galls, attenuate LPS induced cell adhesion molecules expression (ICAM-1 and VCAM-1) in human vein endothelial cells (HUVECs) by blocking AP-1 transcription factor without affecting nuclear transcription factor-ĸβ, thereby inhibiting the adhesion of neutrophils to LPS activated endothelium. In the present study, GCMS analysis of EOPI revealed the presence of Cymene, Borneol, Tetrahydrocarvone, 4-Carvomenthenol, terpinenol, α-Terpinene and β-caryophyllene, Levobornyl acetate, aromadendrene as major constituents in EOPI, which may contribute to the anti-asthmatic effects. β-caryophyllene has been reported to inhibit dextran sulfate sodium induced colitis in mice through CB2 receptor activation and PPARγ pathway. It reduced the disease severity by inhibiting myeloperoxidase and Nacetylglucosaminidase activities and mRNA expression of colonic tumor necrosis factor-α, IL-1β, interferon-γ, and keratinocyte-derived chemokine (Bento et al., 2011). β-caryophyllene also effectively reduced LPS-induced NF-ĸB activation and neutrophil migration in rat paw (Medeiros et al., 2007). Intraperitoneal administration of pcymene ameliorated LPS induced acute lung injury in mice by inhibiting pro-inflammatory cytokines (TNF-α, IL-1β and IL-6) and phosphorylation of IκBα protein and mitogen-activated protein kinases (MAPK) signaling pathway (Xie et al., 2012). In vivo studies of bornyl salicylate reduced neutrophil migration and cytokine release (TNF-α, IL-1β and IL-6) induced by zymosan and fluid leakage induced by acetic acid were also reduced in mice (Vasconcelos et al., 2012). In vitro studies of LPS activated monocytes revealed inhibition of TNF-α, IL-1β, IL-8, IL-10, PGE2 reduction (Hart et al., 2000) and bornyl salicylate reduced NO production in macrophages (Vasconcelos et al., 2012). It could be hypothesized that inhibition of NF-ĸβ pathway by different chemicals present in Pistacia integerrima could be a plausible mechanism for action of EOPI's anti-inflammatory potential. Histopathology findings were in line with our data. The lungs of rats treated with LPS presented severe infiltration of inflammatory cells (majority were neutrophils) and edema. There were hyperplasia bronchial associated lymphoid tissue and proliferation fibrous connective tissue in LPS treated rats. EOPI dependent dose reduced all of these inflammatory changes, and thus, ameliorated the inflammatory conditions of bronchial asthma. EOPI was evaluated against airway hyper-responsiveness in guinea pigs. In present investigation EOPI offered a significant protection against ovalbumin induced bronchoconstriction. The ovalbumin mediated bronchoconstriction is dependent on IgG/ IgE antibody titer and thus leads to mast cell degranulation. Ovalbumin inhalation induced severe bronchoconstriction and thus airway resistance in sensitized guinea pigs which peaked at 2–3 min and returned to the basal level after 10 to 15 min of ovalbumin exposure. Very recently, Adusumalli et al. (2013) demonstrated that aqueous extract of Pistacia integerrima Linn 46.50 mg/kg, p.o. for ten days protected guinea pigs from histamine induced bronchoconstriction. However, in the present investigation EOPI showed protection of immune mediated brochoprovocative response. The bronchodilatory activity of essential oil of Pistacia integerrima J.L. Stewart ex Brandis galls obtained in present study can be attributed to terpenoids since these compounds derived from various medicinal plants have been reported to produce such activities.

6. Conclusion To conclude, presence of rich content of phenolic chemical constituents contributes to the antiasthmatic activity of Pistacia integerrima Stewart ex Brandis galls, thus supporting the veracity of the claims made in the traditional literature. In earlier studies only ethyl gallate isolated from ethyl acetate fraction of Pistacia integerrima Linn galls, attenuated LPS induced ICAM-1 and VCAM-

Please cite this article as: Shirole, R.L., et al., Investigation into the mechanism of action of essential oil of Pistacia integerrima for its antiasthmatic activity. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.02.009i

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1 through the modulation of AP-1 activity without affecting NF-ĸβ. Aqueous extract of Pistacia integerrima Linn showed antiasthmatic activity by mesenteric mast cells membrane stabilizing, in vitro antihistaminic activity on isolated guinea pig tracheal chain preparation and only histamine induced bronchoconstriction in guinea pigs. In contrast, this study demonstrates that pretreatment with EOPI, intervened the inflammatory cascade at various levels starting from antioxidant potential, inhibition of 5-lipoxygenase, protection of 48/80 induced mast cell degranulation, inhibition of L-type Cav channel on isolated guinea pig ileum, anti-angiogenic activity, reduced leukocyte infiltration into airway following allergen exposure and decreasing allergen induced airway hyperresponsiveness which may contribute to anti-asthmatic activity of Pistacia integerrima J.L. Stewart ex Brandis.

Acknowledgments We are thankful to Shri. Amrutbhai Modi for providing financial assistance and IIT Bombay for GCMS analysis of essential oil of Pistacia integerrima J.L. Stewart ex Brandis galls.

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