Indian Journal of Biochemistry & Biophysics Vol. 54, June-August 2017, pp. 140-149

Anti-inflammatory effect of alcoholic extract of pursaetha DC in LPS-induced inflammation in mice and RAW264.7 cells

Rashmi Rekha Kumari1, Madhu Cholenahalli Lingaraju1, Gaurav Gupta1, Amar Sunil More1, Venkanna Balaganur1, Dhirendra Kumar1, Pankaj Kumar2, Dinesh Kumar1, Anil Kuma Sharma3, Santosh Kumar Mishra1 & Surendra Kumar Tandan1* 1Division of Pharmacology and Toxicology; 2Division of Medicine; 3Division of Pathology, Indian Veterinary Research Institute, Izatnagar, Bareilly- 243 122, Uttar Pradesh, India

Received 16 June 2016; revised 24 August 2016

Entada pursaetha () is an active, oriental herbal medicine used to treat various inflammatory diseases. Limited information on the use of stem in ethno medicine in painful conditions of joints prompted us to verify its usage for in vitro and in vivo anti-inflammatory action. The present study was designed to evaluate the anti-inflammatory effects of ethanolic extract of E. pursaetha (EPE) on the production of inflammatory mediators in macrophages and LPS (lipopolysaccharide)- induced systemic inflammation. EPE was studied for its anti-inflammatory activity in on the production of inflammatory mediators in systemic inflammation triggered by LPS and LPS-stimulated RAW 264.7 cells. Plasma tumor necrosis factor alpha (TNF-α), interleukin 1 beta (IL-1β) proteins and total nitrate and nitrite levels were significantly increased in vehicle- treated control group of mice challenged with LPS (4 mg/kg b.wt.). Pre treatment of EPE at 300 mg/kg b.wt. Continuously for 3-days prior to LPS challenge significantly reduced these cytokine levels and nitrate plus nitrite levels in plasma. EPE treatment also significantly inhibited the LPS-induced production of NO (nitric oxide), IL-1β and TNF-α in RAW 264.7 cell. This study demonstrates the anti-inflammatory effects of EPE in both in vivo and in vitro model of inflammation potentially justifying the usage of Entada pursaetha as traditional herbal medicine for the treatment of inflammatory diseases.

Keywords: Anti-inflammatory activity, Cytokines, Entada pursaetha, Lipopolysaccharide, RAW 264.7 cells

Inflammation, a central feature of many different pathways in various cells (macrophages, pathophysiological conditions, occurs in response to Kuppfer cells and hepatocytes)6 and induces nitric tissue injury and is part of the host defense against oxide synthase (iNOS) as well as the production of invading microbes. Macrophages are major immune pro-inflammatory agents, including cytokines (IL-1β, cells that act as the first line of defense against TNF-α, and IL-6), prostaglandins and NO7. On the invading agents, and they respond to pathogen attack other hand, activated macrophages also produce by release of ROS (reactive oxygen species) and anti-inflammatory cytokines such as IL-10 and IL-13, NO, known as cellular signaling molecules and which are used to slow down and terminate the antimicrobial agents1. However, an exaggerated inflammatory response8. Many pathways have been production of inflammatory mediators by macrophage described to explain the effect of anti-inflammatory defensive systems can cause damage to the host2. compounds on macrophages. A number of studies Macrophages are, for example, important contributors reported that plant-derived extracts or plant derivatives to the manifestation of allergic inflammation3 and also such as phenolic compounds and flavonoids show involved in TNF-α4 and IL-1β production in LPS- anti-inflammatory activity by controlling the levels of stimulated macrophages5. During infection LPS acts various inflammatory cytokines or inflammatory on cellular toll like receptor 4 (TLR4), activates mediators including IL-1, IL-6, IL-10, TNF-α, NF-κB, ————— NO, iNOS and COX-2. Moreover, many crude *Correspondence: extracts and chemical constituents of have Fax: +91-581-2303284 pharmacologic effects and clinical benefits. However, E-mail: [email protected] the claims of benefits of many plants or plant based Abbreviations: EPE, Ethanolic extract of E. pursaetha; LPS, medicines marketed to the general population are only Lipopolysaccharide; TNF-α, Tumor necrosis factor alpha; 9 IL, Interleukin; NO, Nitric oxide; ROS, Reactive oxygen species supported by empirical or preliminary scientific data . KUMARI et al.: ANTI-INFLAMMATORY EFFECT OF ENTADA PURSAETHA 141

Entada pursaetha, (Synonyms: E. rheedii, Orissa, India and authenticated by Dr. BN Pandey, E. phaseloides, E. scandens) a very large gigantic Department of Botany, Bareilly College, Bareilly, woody climbing shrub (), is widely distributed in India. A voucher specimen (023/09) was deposited tropical Africa, India, China, Philippines, Guam and in the Indigenous Drug Laboratory of Division of Northern Australia10. In India, it is found in the damp Pharmacology & Toxicology. The powdered stem of forest of Bengal, Bihar, and Orissa, in the forest was refluxed twice with 85% ethanol at 95°C for 8 h. region of eastern and Western Ghats and hilly forest Ethanol was removed under vacuum, and the solid tract of the northern district of Bengal and Deccan11. extract of E. pursaetha stem was hereafter referred as This species can be used as narcotic or as a tonic, etc. “EPE”. The yield of the extract was 8.4% with or used in curing liver troubles, allaying body pains, reference to the dry starting material. EPE was used in warding off cold, curing eye diseases, arthritis, and by suspending in a maximum of 0.2% Polysorbate 80. paralysis12. Mainly seeds, leaves, and stem or stem Doses of EPE 30, 100 and 300 mg/kg body weight bark of E. pursaetha are used for different medicinal were used, based on the earlier work carried out in the purposes. Seed- hepatic complaints, paste applied laboratory (Unpublished data). over affected or inflamed swelling to reduce pain; In our earlier study, various phytoconstituents were leaf-anthelmintic, antiemetic, jaundice, antiseptic, determined in EPE, and the concentration of saponin infantile cold; stem-antiemetic, in diarrhea and skin (glucoside saponin) was highest followed closely by diseases by various tribal communities of India total phenols; total concentration of tannins and in ethno botanical surveys13-15. Recently, the anti- flavonoids was almost similar19. inflammatory effects of Entada pursaetha and its 16,17 In vitro anti-inflammatory activity of EPE in LPS-induced synonym E. phasioloides seed extract in vitro and macrophage cell line in vivo have been reported18. In fact, our recently conducted study demonstrated Propagation and maintenance of cell line - Subculture beneficial effects of Entada pursaetha stem extract in The murine macrophage cell line (RAW 264.7) 19 was procured from NCCS, Pune. On the receipt of experimental knee model of OA and inflammatory 2 bowel disease20. However, the effects of the ethanolic monolayer containing 25 cm flask, cells were extract of E. pursaetha (EPE) of stem on inflammatory subcultured as soon as they reached confluency. disease are yet to be established. In this study we The cells were subcultured using growth medium investigated the effects of EPE on the production of Dulbecco’s modified Eagle’s medium (DMEM) inflammatory mediators by LPS-stimulated RAW with 4mM 264.7 cells. In this study, to verify our hypothesis that L-glutamine adjusted to contain 1.5 g/L sodium EPE could prevent systemic inflammation triggered bicarbonate and 4.5 g/L glucose and supplemented by LPS in mice, we demonstrated the effect of with 10% (v/v) fetal calf serum and antibiotic Entada pursaetha stem on the release of inflammatory (100 U/mL penicillin, 100 μg/mL of streptomycin). mediators both in vitro and in vivo. The growth condition was 37°C temperature and a humidified atmosphere containing 5% CO2 condition. Materials and Methods The subculture was done in 10% fetal calf serum containing DMEM medium with the help of Animals cell scraper. Male albino mice (Livestock Resource Section, First old medium was removed. Secondly, fresh Indian Veterinary Research Institute) weighing 20-25 g medium was added, thirdly, cells were dislodged were used. They were housed at a maximum of using cell scraper and dispensed into a new flask. four per cage on a 12 h day/night cycle at a temperature Flasks were then kept in an incubator with the slightly of 22 ± 1°C. Water and food were provided loose cap. The volume of medium used for subculture ad libitum. All animal experimental procedures were was according to the usual ratio of medium volume to duly approved (IAEC/2009/011) by Animals Ethics surface area, i.e. 0.2-0.5 mL/cm2. The cells were Committee of Indian Veterinary Research Institute. subcultured as soon as they reached confluency and Reagents and materials maintained in 2% fetal calf serum containing medium

Preparation of alcoholic extract of E. pursaetha according to our experimental need. Whenever The mature stem of E. pursaetha was brought from needed, the number of flasks were increased by the jungles of Bhawanipatna, District- Kalahandi, subculture and maintained in 2% DMEM, changed 142 INDIAN J. BIOCHEM. BIOPHYS., VOL. 54, JUNE-AUGUST 2017

3 times per week. Most cell stop growing as the pH value of the content of four well was used as cell falls from 7.0-6.5 and starts to lose viability between viability expression as % of control. The assay was 6.5 and 6.0. So if medium goes from red through performed in triplicate. orange to yellow, the medium should be changed. Based on above exploratory work, three increasing If there is a rapid change in pH, the medium must be non-cytotoxic concentration levels of EPE 20, 30 and changed frequently, then subculture must be done and 50 μg/mL were selected for assessing the effect of seeding density must be reduced. these concentrations of EPE on NO, IL-1β, TNF-α level in medium LPS-stimulated macrophage cell. For Preparation of stock solution of ‘EPE’ each assay, there were six groups I-VI where I was EPE was suspended in DMSO at concentration of Naïve control, II was vehicle control and treated with 100 mg/mL (0.1% DMSO final concentration). LPS at 2 μg/mL (final concentration in well) only, Different concentrations of EPE were prepared whereas III, IV, V were treated with LPS (2 μg/mL) from this stock solution in 2% DMEM. Then filter and 20, 30 and 50 μg/mL concentration of sterilized. For NO estimation all concentration were EPE respectively. Group VI treated with LPS prepared in phenol red free DMEM. and dexamethasone (0.5 μg/mL) and acted as standard/reference treatment group. Preparation of stock concentration of LPS A stock solution of LPS (Escherichia coli 055: B5, Nitric oxide assay Sigma-Aldrich) @ 200 μg/mL in PBS, pH 7.4 was For nitric oxide assay, phenol red free DMEM prepared and then filter sterilized. [The composition medium was used for seeding step onward. Cells were 4 of PBS was Na2HPO4 (1.14 g) or Na2HPO4.2H2O seeded in 96 well plates at a concentration of 5 × 10 (1.44 g), KCl (0.2 g), KH2PO4 (0.2 g), NaCl (8 g) per in 10% phenol red free DMEM medium and liter of sterilized distilled water]. incubated for 12 h. After removing medium, a cell of group II, III, IV were treated with phenol red free 2% MTT Assay for cell viability DMEM containing 20, 30 and 50 μg/mL EPE MTT (3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl while group VI was treated with dexamethasone at tetrazolium bromide) assay for cell viability was 0.5 μg/mL whereas group I was control. Now Cells of done. MTT, a yellow color dye converted by living all groups except control were treated with 10 μL cell with active mitochondria into blue color formazan of a stock solution of LPS. After incubation for 24 h, crystals. MTT assay was performed to determine non- supernatant (medium) was collected and stored at cytotoxic concentrations of EPE to be employed for −80°C for estimation of nitric oxide. Nitric oxide the further study. The different concentrations of EPE concentration was determined by the method were prepared from a stock solution in 2% DMEM. described by Sastry et al.21. The details of this These concentrations were 20, 50, 100, 200 and methodology are furnished in the estimation of 400 μg/mL and vehicle control group was treated nitrite and nitrate in plasma. Results are expressed as with 0.1% DMSO containing 2% DMEM. Cells mean ± SE of three replicates of one representative (RAW264.7) were seeded in 10% DMEM at 6 experiment. required concentration (2-5 × 10 /mL, 100 μL volume) in 96-well plates and incubated for 12 h. After Cytokines: TNF-α and IL-1β assay preconditioning, media was decanted and 100 μL of Cells were seeded in 96-well plate at a 2% fresh media containing different concentrations of concentration of 5 × 104 in 10% DMEM medium and EPE was added to wells. Vehicle control cells were incubated for 12 h. After removing medium, cells of incubated with 0.1% DMSO. After 48 h of incubation, group II, III, IV were treated with 2% DMEM 20 μL of MTT dye (5 mg MTT/mL of PBS) was containing 20, 30 and 50 μg/mL EPE while cells added in each well and incubated for 4 h in the dark at of group VI were treated with dexamethasone at 37°C and 5% CO2. Further media was removed by 0.5 μg/mL whereas group I was control. Macrophage tilting the plate from a side, DMSO (100 μL) was Cells of all groups except control were treated with added to each well, formazan crystals formed by 10 μL of stock solution of LPS. After incubation for viable cells. A violet blue colour develops in wells. 24 h, cell supernatant will be harvested and stored at Absorbance was taken at 550 nm in ELISA reader −80°C until tested for cytokines. ELISA estimated the (Spectra Max190, Molecular devices). The mean OD quantities of these cytokines following manufacturer’s KUMARI et al.: ANTI-INFLAMMATORY EFFECT OF ENTADA PURSAETHA 143

instruction. Results were expressed as mean ± SE of In this procedure, 100 μL of plasma or standard three replicates of one representative experiment. (potassium nitrate) was added to test tubes (12 × 75 mm) to which 400 μL of carbonate buffer was added, Determination of anti-inflammatory activity of EPE using followed by small amount (~150 mg) of cadmium in vivo model of inflammation in mice filings (washed in the same buffer and dried on a Lipopolysaccharide (LPS)-induced lethality study filter paper). The tubes were then incubated at To test if EPE extract can increase survival time in room temperature for 1h with thorough shaking. The a mouse model of sepsis, mice were injected with a reaction was stopped by 400 μL of 120 mM zinc single dose of vehicle or extract before LPS challenge sulfate solution under vortex, and the solution was (15 mg/kg IP, E. coli 055: B5). The dose of LPS allowed to stand for 10 min. The tubes were then was close to LD95 and dose of extract (30, 100 and centrifuged at 400 g for 10 min. 300 mg/kg) were based on earlier studies in the Aliquots (150 μL) of the clear supernatant laboratory. Animals were observed for three days for were transferred to the wells of a microplate mortality. The groups were as follows: (96 well), in triplicate. Then, components of the

Effect of EPE on Pro-inflammatory cytokines TNF-α, IL-1β, Griess reagent were added with gentle mixing each and nitric oxide levels in LPS-induced systemic inflammation time, 75 μL of 1.0 % sulfanilamide and 75 μL of 1.0% Systemic inflammation was induced by a single N-(1-Naphthyl) ethylenediamine. After 10 min, the dose of LPS (4 mg/kg) by intra-peritoneal injection of absorbance was measured at 545 nm against a blank saline. Mice were divided into five groups of six containing no biological sample in a microplate reader animals each. Mice in group I served as naive control (Spectra Max190, Molecular devices) using the and received normal saline.Mice in group II were path check option. By using standard curve the administered vehicle orally while mice in group III, amount of nitrate and nitrite present in the sample IV and V were administered EPE orally at 30, 100 was calculated. and 300 mg/kg suspended in the vehicle for three days (Table 1). On the 3rd day after 3h of vehicle or EPE Histopathology administration, animals from group II III, IV and V After collecting blood as above, animals were were challenged with LPS (4 mg/kg, i.p.). Blood from sacrificed by cervical dislocation, liver and lung were mice of all groups was collected in heparinised tube removed and fixed in 10% buffered formalin for by cardiac puncture, 90 min after LPS administration. histopathological studies (H&E Staining).

Separation of plasma was done in a refrigerated Statistical analysis centrifuge tube at 7000 rpm for 10 min and stored Data were expressed as mean ± SE. Statistical at −80°C until analyzed for parameters: TNF-α and analysis of data was performed using GraphPad prism IL-1β (EIA kit, E-Bioscience) and Nitrate-nitrite (NOx). 4. Data were analyzed by ANOVA and, means

Estimation of nitrate-nitrite (NOX) were compared with Tukey’s multiple comparison Nitrate and nitrite in the plasma were estimated tests. A minimum value of P <0.05 was considered following the procedure described Sastry et al.21. statistically significant. The principle of the assay is the conversion of nitrate to nitrite by copper-cadmium alloy and then color Results development by Griess reagent (Sulfanilamide and In vitro anti-inflammatory activity of EPE in LPS-induced N-(1-Naphthyl) ethylenediamine) in acidic medium. macrophage cell line The objective of study was to measure effect of Table 1 — Animal grouping and drug administration EPE on pro-inflammatory mediators such as TNF-α, (6 mice in each group) IL-1β proteins and NO production in LPS -stimulated Group Treatment EPE (orally 3h LPS macrophage cell line RAW264.7 cells. (n=6) before LPS) (LD95 IP) I Control – MTT assay Vehicle + In the first place, an MTT assay for cell viability II – 15 mg/kg b. wt LPS was performed to determine three non-cytotoxic III EPE 1+ LPS 30 mg/kg b. wt 15 mg/kg b. wt concentrations of EPE. The result of MTT cell IV EPE 2+ LPS 100 mg/kg b. wt 15 mg/kg b. wt viability had been depicted in (Table 2). MTT is a V EPE3+ LPS 300 mg/kg b. wt 15 mg/kg b. wt yellow dye which is converted into blue formazan 144 INDIAN J. BIOCHEM. BIOPHYS., VOL. 54, JUNE-AUGUST 2017

crystals by live cells. Result of cell survival was was significantly decreased (P <0.05) than that in expressed in percent of control cells with no EPE single LPS stimulation (P <0.01). Furthermore, the treatment. The cell viability was 107.4 and 91.95% more increasing on dosage of the extract, the better with 20 and 50 μg/mL concentrations of EPE treatment effects of antagonizing pro-inflammatory cytokines which were statistically nonsignificant when compared IL-1β was noted which showed dose-dependent with control cells with no EPE treatment. Cell relation between anti-inflammatory effect and EPE viability at 100 μg/mL EPE concentrations was concentrations. IL-1β level was brought to normal in 60.94% of control which was significantly less LPS stimulated RAW264.7 cells treated with EPE at a (P <0.05) than control with no EPE. Further with concentration of 30, 50 μg/mL and dexamethasone. 200 μg/mL and 400 μg/mL EPE concentrations, cell At a 20 μg/mL concentration of EPE, IL-1β level was viability was 8.67 and 11.59%, respectively. EPE has significantly less (P <0.05) than cells stimulated with cytotoxic effect at 100, 200 and 400 μg/mL and non- LPS only but not brought to control value of cytotoxic at 20 and 50 μg/mL concentrations. unstimulated cells. Therefore, 20, 30 and 50 μg/mL EPE concentrations NO: The concentration of NO was indirectly were used for further study for cytokine assay and measured by measuring total nitrate-nitrite NO levels. concentration in culture medium. Nitrate-Nitrite level in medium was significantly higher (P <0.05) when Effect of EPE on pro-inflammatory cytokines of nitric oxide cells were stimulated with LPS as compared to The result of TNF-α, IL-1β proteins and NO unstimulated cells (Table 3). This increase in levels in culture medium at different concentrations NO level was reduced in concentration- dependent of EPE had been presented in Table 3. manner in EPE-treated cells but not brought to TNF-α: TNF-α levels in culture medium was unstimulated cells value. Dexamethasone treatment significantly increased (P <0.05) in LPS stimulated also did not bring the value to unstimulated cells as compared to unstimulated cells (Table 3). cells level. EPE at concentration of 30 and 50 μg/mL significantly reduced (P <0.05) the TNF-α level in Determination of anti- inflammatory activity of EPE using culture medium as compared LPS- stimulated cells. in vivo model of inflammation in mice

EPE at a concentration of 20 μg/mL did not Lipopolysaccharide (LPS) lethality study significantly reduce TNF-α level in LPS- stimulated There was no significant reduction in mortality RAW264.7 cells. in EPE-treated groups as compared with vehicle- IL-1β: As shown in Table 3, after LPS stimulation, treated groups in LPS-induced sepsis in mice (results with the extract intervention for 24h, secretion of IL-1β not presented).

Table 2 — MTT assay for cell viability. Values are expressed as means ± SE. EPE concentration (µg/mL) 0 20 50 100 200 400 Cell survival (% of control) 100.0 ± 0.00a 107.4 ± 2.765a 91.95±3.48a 60.94 ± 16.4b 8.763 ± 1.56c 11.59 ± 1.382c Result shown is atypical result of three independent experiments with similar results. Cell survival is expressed as % of control. Value with dissimilar superscript differs significantly.

Table 3 — Effects of EPE on LPS-induced production of pro-inflammatory cytokine and nitric oxide in RAW 264.7 cells. Values are expressed as mean ± SE. Treatments TNF-α IL-1β Total nitrate & (pg/mL ) (pg/mL ) nitrite (nM/mL ) Control 8.635±1.131a 8.630±1.662a 22.03±2.275a LPS (2 µg/mL) 104.8±21.54b 975.7±40.86b 106.3±1.289b LPS+EPE (20 µg/mL) 97.83±11.09b 449.3±24.77c 99.10±1.249c LPS+EPE (30 µg/mL) 50.61±11.08a 77.84±5.918a 89.11±0.5888d LPS+EPE (50 µg/mL) 26.51±2.663a 61.95±4.946a 79.02±0.8329e LPS+DEXA (0.5 µg/mL) 9.653±1.410a 31.05±3.800a 49.48±0.3543f Three independent experiments were performed. Value with dissimilar superscript differs significantly. KUMARI et al.: ANTI-INFLAMMATORY EFFECT OF ENTADA PURSAETHA 145

Table 4 — Effects of EPE on inflammatory cytokines TNF-α, IL-1β in LPS-induced systemic inflammation (4 mg/kg b.wt., i.p.) in micen Treatments TNF-α IL-1β Total nitrate & (pg/mL) (pg/mL) nitrite (nM/mL) Naïve control 19.90±0.4095a 41.76±1.904a 8.106±0.2562a Vehicle control 38.91±0.6185b 72.71±1.627b 20.26±1.583b EPE@ 30 mg/kg 39.63±0.7010b 74.27±1.986b 21.01±0.9589b EPE@ 100 mg/kg 38.39±0.8145b 75.58±1.558b 17.44±1.808b EPE@ 300 mg/kg 32.24±0.5897c 54.79±1.948a 8.438±0.2595a n=6; Value with dissimilar superscript differ significantly.

Fig. 1 — Representative sections of liver of naive control group showing normal histological features H&E, 10X (A), LPS challenged (4 mg/kg b.wt.) vehicle - treated control group of mice showing various histopathological features; Infiltration of leucocytes in portal area, H&E, 10X (B), mice pretreated with EPE (300 mg/kg b.wt.) showing less leucocyte infiltration in portal area and sinusoid, H&E, 10 X (C).

Fig. 2 — Representative sections of lung of naive control group showing normal histological features H&E, 10X (A), LPS challenged (4 mg/kg b.wt.) vehicle - treated control group of mice showing various histopathological features; Infiltration of leucocytes in bronchiole as well as interalveolar septae, H&E, 10 X (B), mice pretreated with EPE (300 mg/kg b.wt.) showing less leucocyte infiltration in decreased number of neutrophils in alveoli and almost normal structure of lung parenchyma, H&E, 10 X (C). Effect of EPE on Pro-inflammatory cytokines IL-1β, TNF-α and attenuated by pretreatment of EPE at the rate of nitric oxide levels in systemic inflammation 300 mg/kg body wt. Results of plasma levels of cytokines IL-1β, TNF-α proteins and nitric oxide are summarized in Table 4 Histopathology following systemic inflammation induced by a single In the naive control group, light microscopy dose of LPS (4 mg/kg). Plasma TNF-α and IL-1β showed no infiltration of polymorph nuclear cells levels were significantly increased (P <0.05) in (PMNs) in the liver (Fig. 1A) or lungs (Fig. 2A). In vehicle-treated control group of mice challenged with contrast, increased infiltration of PMNs in both liver LPS. Pretreatment of EPE at 300 mg/kg body wt. (Fig. 1B) and lungs (Fig. 2B) was noted at 90 min continuously for 3 days prior to LPS challenge after injection of LPS. In mice pretreated with significantly reduced (P <0.05) these cytokine levels EPE, infiltration of PMNs in liver (Fig. 1C) and lungs in plasma. (Fig. 2C) was reduced. Results also showed that LPS challenge to mice caused significant increase in nitrate and nitrite Discussion plasma levels as compared to naive control group Plant products have been historically important in of mice and this increase was significantly (P <0.05) the prevention and treatment of illness. Plants are a 146 INDIAN J. BIOCHEM. BIOPHYS., VOL. 54, JUNE-AUGUST 2017

rich source of active ingredients for health care effectively decreased NO production possibly via products with many blockbuster drugs being suppressing the mRNA and protein expressions of directly or indirectly derived from plants22. The iNOS in LPS-stimulated RAW264.7 cells. Moreover, pharmacological activities of many high-value plant- the inhibitory effects on the LPS-induced expressions derived natural products remain undiscovered or were not due to the cytotoxicity of EPE as assessed by unexplored. We used ethanolic extract of E. pursaetha MTT assay. (EPE) for exploring its anti-inflammatory activity. TNF-α and IL-1β proteins are major pro- E. pursaetha is an active, oriental herbal medicine inflammatory cytokines in various immune cells used to treat various inflammatory diseases. In spite such as macrophages, monocytes and T cells and of its famous legacy, the pharmacological effects have various pro inflammatory effects in chronic have not been fully explored. The present study was inflammatory diseases25,32. In this study, EPE undertaken to elucidate the pharmacological and effectively inhibited the production of these two biological effects of EPE on the production of cytokines by possibly suppressing their mRNA inflammatory mediators in macrophages and LPS- expressions in LPS-stimulated RAW 264.7 cells. The induced systemic inflammation. results of this study demonstrate that EPE inhibited We showed that EPE suppressed the production of the generation of pro-inflammatory cytokines in a TNF-α, IL-1β proteins and NO in LPS-stimulated concentration-dependent manner that is paramount in RAW264.7 cells which are primary peritoneal the generation of an inflammatory response in macrophages. Macrophages actively participate in activated macrophages. In our experiments, LPS was inflammatory responses by releasing pro-inflammatory used as the prototypical inflammatory stimulus cytokines (TNF-α and IL-1β) and inflammatory factors because of its ability to initiate a range of (NO and PGE2) that recruit additional immune cells to pro-inflammatory mediators33. LPS, as well as TNF-α sites of infection or tissue injury23. The production of and interferon gamma (IFN-γ), are effective activator these inflammatory molecules by macrophages can be of NF-κB34. NF-κB is known to play a critical role in induced in response to LPS stimulation24. Therefore, regulating cell survival genes and controlling most inhibitors of these inflammatory molecules have been inflammatory responses by induction of inducible considered as a candidate for an anti-inflammatory enzymes (iNOS and COX-2), pro-inflammatory drug25 to alleviate a variety of disorders caused by cytokines (IL- 1, IL-2, IL-6 and TNF-α), chemokines activation of macrophages. (IL-8, MIP-1β, MCP-1 and RANTES), adhesion RAW 264.7 macrophages provide us with an molecules (ICAM, VCAM and E-selectin), growth excellent model for anti-inflammatory drug screening factors, some of the acute phase proteins, and and for subsequently evaluating the inhibitors of the immune receptors34. Therefore, blocking the NF-κB pathways that lead to the induction of pro- transcriptional activity in the macrophage nucleus can inflammatory enzymes and the production of pro- suppress the expression of inflammatory mediators. inflammatory cytokines. EPE has been reported to EPE might have reduced the pro-inflammatory possess anti-inflammatory activity in carrageenan- cytokines and NO by blocking NF-κB. induced inflammation in mice. However, no report Severe sepsis may not be reversible and result has been available on the mode of action involved26. in high mortality. In this study, we established a In this study, we show that EPE acts as an anti- mild systemic inflammation model in which mice inflammatory substance in vitro. NO is a free radical were challenged i.p. by a single dose of LPS35 that plays a pivotal role in cell survival and death and (4 mg/kg b. wt.). According to our preliminary data, plays various pro-inflammatory effects on many cell the LPS-challenged mice were sick from LPS challenge, types27. High levels of NO generated by iNOS in but none died during one week of observation period. inflamed tissue have been shown to be cytotoxic in A sub-lethal endotoxin shock murine model has been studies of many types of inflammatory diseases established. The sub-septic doses of endotoxin LPS including asthma28, arthritis29, and cardiovascular can activate distinct immune responses such as diseases30 leading to cell death31. LPS stimulation systemic inflammation and have been used in significantly increased the concentrations of NO. previous studies for different purposes36,37. Using our Therefore, NO inhibition in inflammation has the established sub-septic (sublethal) animal model, the potential therapeutic implications. We found that EPE effects of EPE pretreatment in vivo on acute systemic KUMARI et al.: ANTI-INFLAMMATORY EFFECT OF ENTADA PURSAETHA 147

inflammation were measured. Our results showed endotoxin-induced sepsis43. Production of NO by that LPS challenge (vehicle control) significantly iNOS is beneficial in fighting bacteria, but its increased plasma cytokine levels of IL-1β or TNF-α overproduction can be harmful as shown in suggesting that systemic inflammation was induced. endotoxic shock44. There is increasing evidence Pretreatment of EPE (300 mg/kg) 3h before systemic that overproduction of TNF-α during infection also inflammation by LPS significantly decreased plasma leads to severe systemic toxicity and even death45. IL-1β and TNF-α level in mice (Table 4). IL-1β and Evidence supporting this hypothesis comes from TNF-α play a critical role in the manifestation of reports indicating that mediators (e.g. TNF-α and systemic inflammation and are believed to be a pro- interleukins) produced by endotoxin challenge can inflammatory and EPE pretreatment before acute induce iNOS expression and produce large amounts systemic inflammation alleviates acute inflammation of NO46. This point was further supported by the status via inhibiting plasma IL-1β and TNF-α results in which EPE reduced plasma nitrate-nitrite production. Macrophages are large mononuclear concentration possibly by reducing iNOS protein phagocytic cells involving innate and adaptive expression in tissues challenged by LPS. immunity in vivo. These are migratory cells and are In systemic inflammation, histopathology section found in most tissues of the body. The releases of of liver and lung (Fig. 1 & 2) also showed IL-1β and TNF-α by macrophages may induce local inflammatory changes such as blood vessel dilatation protective effects but they can have damaging effects, and infiltration of neutrophils in liver and lung when released systemically. parenchyma in LPS challenged animals. But the EPE TNF-α is the target for development of new (300 mg/kg) pretreatment at this dose resulted in medication for disease involving inflammation such less infiltration of leucocytes in liver and lung as rheumatoid arthritis. Inhibition of elevated TNF-α (Figs. 1 & 2). Further, reduced cytokines and NO induced by LPS has been a common practice for plasma levels at this dose in mice were evaluation of anti-inflammatory effect of drug associated with decreased leukocyte infiltration in candidates38,39. Our results showed lower production lung and liver. of TNF-α not only in LPS-stimulated RAW 264.7 Phenolic compounds were detected in high cells pretreated by EPE as stated above but also in amounts in Entada africana47. Qualitative analysis plasma from LPS-challenged mice pretreated with was done with Entada africana crude extract also EPE (300 mg/kg) suggests TNF-α as a target in anti- revealed the presence of polyphenols in this extract48. inflammatory strategy. The biological effects of polyphenols have been Recently, LPS-injected animal models have been reported to be related to their ability to influence introduced to evaluate the effects of medicine, food, cell signaling pathways and to modulate pro and 49,50 or nutrition on systemic inflammation40. It has been anti- inflammatory gene expression . Therefore, proposed that NO production in response to we suggest that the phenolic compounds present in 19 LPS regulates important aspects of septic shock. (41) Entada pursaetha may be responsible for the Therefore, to investigate whether EPE has a protective observed effects on RAW264.7 cells and in vivo effect on LPS-induced systemic inflammation, the inflammatory cytokines and NO in mice. Overall, this plasma nitrite-nitrate levels in LPS-treated mice was study demonstrates the anti-inflammatory effects of examined in this study. It was found that pretreatment EPE in both in vivo and in vitro model of of mice with EPE (300 mg/kg, orally) inhibited inflammation suggesting that Entada pursaetha might plasma nitrite-nitrate production in the LPS have substantial therapeutic potential for treatment of challenged mice (Table 4). This finding further inflammation and inflammatory diseases. supports the hypothesis that EPE has a potent anti- References inflammatory activity in vivo. 1 Blazovics A, Hagymasi K & Pronai L, Cytokines, A large amount of inflammatory mediators prostaglandins, nutritive and non-nuitritive factors in produced in the body is thought to contribute to inflammatory bowel diseases. Orv Hetil, 145 (2004) 2523. the LPS-induced symptoms of septic shock and 2 Huo M, Cui X, Xue J, Chi G, Gao R, Deng X, Guan S, 42 Wei J, Soromou LW, Feng H & Wang D, Anti-inflammatory mortality . Amongst the inflammatory mediators, NO effects of linalool in RAW 264.7 macrophages and is known to be closely associated with hypotension lipopolysaccharide-induced lung injury model. J Surg Res, and hypo responsiveness to vasoconstrictors in 180 (2013) 47. 148 INDIAN J. BIOCHEM. BIOPHYS., VOL. 54, JUNE-AUGUST 2017

3 Varga A, Budai MM, Milesz S, Bacsi A, Tozser J & Benko S, Effect of alcoholic extract of Entada pursaetha DC on Ragweed pollen extract intensifies lipopolysaccharide- monosodium iodoacetate-induced osteoarthritis pain in rats. induced priming of NLRP3 inflammasome in human Indian J Med Res, 141 (2015) 454. macrophages. Immunology, 138 (2013) 392. 20 Gupta G, More AS, Kumari RR, Lingaraju MC, Pathak NN, 4 Haddad JJ & Land SC, Redox/ROS regulation of Kumar D, Kumar D, Mishra SK, Tandan SK, Protective lipopolysaccharide-induced mitogen-activated protein kinase effect of alcoholic extract of stem of Entada pursaetha (MAPK) activation and MAPK-mediated TNF-α biosynthesis. in dextran sulphate sodium-induced colitis in mice. Br J Pharmacol, 135 (2002) 520. Indian J Biochem Biophys, 52 (2015) 147. 5 Ryan KA, Smith MF Jr, Sanders MK & Ernst PB, Reactive 21 Sastry KVH, Moudgal RP, Mohan J, Tyagi JS & Rao GS, oxygen and nitrogen species differentially regulate Toll- Spectrophotometric determination of serum nitrite and nitrate like receptor 4-mediated activation of NF-kappa B and by copper-cadmium alloy. Analytical Biochem, 306 (2002) 79. interleukin-8 expression. Infect Immun, 72 (2004) 2123. 22 Rishton GM, Natural products as a robust source of new 6 Chen YC, Shen SC, Chen LG, Lee TJ & Yang LL, Wogonin, drugs and drug leads: past successes and present day issues. baicalin, and baicalein inhibition of inducible nitric oxide Am J Cardiol, 101 (2008) 43. synthase and cyclooxygenase-2 gene expressions induced by 23 Boscá L, Zeini M, Través PG & Hortelano S, Nitric oxide nitric oxide synthase inhibitors and lipopolysaccharide. and cell viability in inflammatory cells: a role for NO in Biochem Pharmacol, 61 (2001) 1417. macrophage function and fate. Toxicology, 208 (2005) 249. 7 Glushkova OV, Parfenyuk SB, Khrenov MO, Novoselova TV, 24 Ajizian SJ, English BK & Meals EA, Specific inhibitors of Lunin SM, Fesenko EE & Novoselova EG, Inhibitors of p38 and extracellular signal-regulated kinase and mitogen- TLR-4, NF-κB, and SAPK/JNK signaling reduce the activated protein kinase pathways block inducible nitric toxic effect of lipopolysaccharide on RAW 264.7 cells. oxide synthase and tumor necrosis factor accumulation in J Immunotoxicol, 10 (2013) 133. murine macrophages stimulated with lipopolysaccharide and 8 Abuarqoub H, Foresti R, Green CJ & Motterlini R, interferon-gamma. J Infect Dis, 179 (1999) 939. Heme oxygenase-1 mediates the anti-inflammatory effect of 25 Andreakos E, Foxwell B & Feldmann M, Is targeting Curcumin within LPS-stimulated human monocytes. Am J Toll-like receptors and their signaling pathway a useful Physiol Cell Physiol, 290 (2006) 1092. therapeutic approach to modulating cytokine-driven 9 Koretz RL & Rotblatt M, Complementary and alternative inflammation? Immunol Rev 202 (2004) 250. medicine in gastroenterology: the good, the bad, and the 26 Kalpanadevi V, Shanmugasundaram R & Mohan V, ugly. Clin Gastroenterol Hepatol, 2 (2004) 957. Anti-inflammatory activity of seed extract of Entada 10 Brenan JPM, Notes on . Kew Bulletin, pursaetha DC against carrageenan induced paw edema. Sci 2 (1955) 161. Res Rep, 2 (2012) 69. 11 Chopra RN, Chopra IC, Handa KL & Kapur LD, 27 Garcia X & Stein F, Nitric oxide. Semin Pediatr Infect Dis, Entada pursaetha, In: Chopra’s Indigenous drug of India, 17 (2006) 55. First edition reprint (Academic Publisher, Kolkata), 2006, 334. 28 Barnes PJ & Liew FY, Nitric oxide and asthmatic 12 Chopra RN, Nayar SL & Chopra IC, Glossary of Indian inflammation. Immunol Today, 16 (1995) 128. medicinal plants. (CSIR, New Delhi), 1956 107. 29 Abramson SB, Amin AR, Clancy RM & Attur M, The role 13 Padal SB & Satya vathi K, Phytomedicinal flora and of nitric oxide in tissue destruction. Best Pract Res Clin their folk claim of Visakhapatnam district agency, Rheumatol, 15 (2001) 831. Andhra Pradesh, (India). IOSR-JESTFT, 3 (2013) 9. 30 Llorens S & Nava E, Cardiovascular diseases and the nitric 14 Paul S, Devi N & Sarma GC, Medicinal plants of Ultapani oxide pathway. Curr Vasc Pharmacol, 1 (2003) 335. forest range under Holtugaon division, Manas biosphere 31 Tripathi P, Tripathi P, Kashyap L & Singh V, The role of reserve (Assam). IJABPT, 2 (2011) 257. nitric oxide in inflammatory reactions. FEMS Immunol Med 15 Sai Vishnu Priya K & Srinivasa Rao JV, Exploration of tribal Microbiol, 51 (2007) 443. knowledge of Entada pursaetha DC: An endangered gigantic 32 Bondeson J, The mechanisms of action of disease-modifying medicinal legume in Eastern ghats. Ethnobotl Leaflets, antirheumatic drugs: a review with emphasis on macrophage 12 (2008) 36. signal transduction and the induction of proinflammatory 16 Bhogireddy N, Mathi P, Gaddaguti V, Ambatipudi N & cytokines. Gen Pharmacol, 29 (1997) 127. Bokka VR, Evaluation of the in vitro anti-inflammatory 33 Abreu MT & Arditi M, Innate immunity and toll-like activity of the ethanol extract from the seeds of receptors: clinical implications of basic science research. Entada pursaetha DC. Int J Pharm Bio Sci, 7 (2016) 546. J Pediatr, 144 (2004) 421. 17 Bhogireddy N, Mathi V, Ambatipudi N, Talluri V & 34 Hanada T & Yoshimura A, Regulation of cytokine Bokka VR, In vitro antiinflammatory and biofractionation signaling and inflammation. Cytokine Growth Factor Rev, of Entada pursaetha DC ethanol seed extract in LPS 13 (2002) 413. induced RAW 264.7 macrophage cells. Advan Biol Res, 35 Johnson JL, Hong H, Monfregola J & Catz SD, Increased 9 (2015) 109. survival and reduced neutrophil infiltration of the liver in 18 Dawane JS, Pandit VA & Rajopadhye BD, Experimental Rab27a- but not Munc13-4-deficient mice in lipopoly- evaluation of anti-inflammatory effect of topical application saccharide-induced systemic inflammation. Infect Immun, of Entada phaseoloides seeds as paste and ointment. 79 (2011) 3607. North Am J Med Sci, 3 (2011) 513. 36 De Maio A, Torres MB & Reeves RH, Genetic determinants 19 Kumari RR, More AS, Gupta G, Lingaraju MC, Balaganur V, influencing the response to injury, inflammation, and sepsis. Kumar P, Kumar D, Sharma AK, Mishra SK & Tandan SK, Shock 23 (2005) 11. KUMARI et al.: ANTI-INFLAMMATORY EFFECT OF ENTADA PURSAETHA 149

37 Xia Y & Krukoff TL, Cardiovascular responses to subseptic 44 Nathan C, Nitric oxide as a secretory product of mammalian doses of endotoxin contribute to differential neuronal cells. FASEB J, 6 (1992) 3051. activation in rat brain. Mol Brain Res, 89 (2001) 71. 45 Langrehr JM, Hoffman RA, Lancaster JR & Simmons RL, 38 Wang Q, Zhang Y, Hall JP, Lin LL, Raut U, Mollova N, Nitric oxide-a new endogenous immunomodulator. Green N, Cuozzo J, Chesley S, Xu X, Levin JI & Patel VS, Transplantation, 55 (1993) 1205. A rat pharmacokinetic/pharmacodynamic model for assessment 46 Szabo C, Wu CC, Gross SS, Thiemermann C & Vane JR, of lipopolysaccharide-induced tumor necrosis factor-alpha Interleukin-1 contributes to the induction of nitric oxide synthase production. J Pharmacol Toxicol Methods, 56 (2007) 67. by endotoxin in vivo. Eur J Pharmacol, 250 (1993) 157. 39 Wu M & Gu Z, Screening of bioactive compounds 47 Karou SD, Tchacondo T, Ouattara L, Anani K, Savadogo A, from moutan cortex and their anti-inflammatory activities Agbonon A, Attaia MB, de Souza C, Sakly M & Simpore J, in rat synoviocytes. Evid Based Complement Alternat Med, Antimicrobial, antiplasmodial, haemolytic and antioxidant 6 (2009) 57. activities of crude extracts from three selected Togolese 40 Oberbeck R, Schmitz D, Wilsenack K, Schuler M, Biskup C, medicinal plants. Asian Pac J Trop Med, 4 (2011) 808. Schedlowski M, Nast-Kolb D & Exton MS, Prolactin 48 Njayou FN, Aboudi ECE, Tandjang MK, Tchana AK, modulates survival and cellular immune functions in septic Ngadjui BT & Moundipa PF, Hepatoprotective and mice. J Surg Res, 113 (2003) 248. antioxidant activities of stem bark extract of Khaya 41 MacMicking JD, Nathan C, Hom G, Chartrain N, grandifoliola (Welw) CDC and Entada africana Guill. et Fletcher DS, Trumbauer M, Stevens K, Xie QW, Sokol K, Perr. J Nat Prod, 6 (2013) 73. Hutchinson N, Chen H & Mudgett JS, Altered responses 49 Lacour S, Antonios D, Gautier JC & Pallardy M, to bacterial infection and endotoxic shock in mice lacking Acetaminophen and lipopolysaccharide act in synergy for inducible nitric oxide synthase. Cell, 81 (1995) 641. the production of pro-inflammatory cytokines in murine 42 Ando H, Takamura T, Ota T, Nagai Y & Kobayashi K, RAW264.7 macrophages. J Immunotoxicol, 6 (2009) 84. Cerivastatin improves survival of mice with 50 Tominari T, Hirata M, Matsumoto C, Inada M & Miyaura C, lipopolysaccharide-induced sepsis. J Pharmacol Exp Ther, Polymethoxy flavonoids, nobiletin and tangeretin, prevent 294 (2000) 1043. lipopolysaccharide-induced inflammatory bone loss in 43 Thiemermann C, Nitric oxide and septic shock. Gen Pharmacol, an experimental model for periodontitis. J Pharmacol Sci, 29 (1997) 159. 119 (2012) 390.