Toxicological Evaluation of Emblica officinalis Fruit Extract and its Anti-inflammatory and Free Radical Scavenging Properties

ISSN : 0973-1296 October-December 2015 | Volume 11 | Issue 44 (Supplement 3) Included Pharmacognosy Magazine • Volume 11 • Issue 44 (Supplement 3) • October-December 2014 • Pages ***-*** Pharmacognosy Magazine Publication of Pharmacognosy Network Worldwide www.phcog.com Impact Factor® for 2012: 1.525 Phcog.Net - Bringing Medicinal Plant Researchers Together CAB Abstracts, Caspur, Chemical Abstracts, CNKI (China National Knowledge Infrastructure), CSA databases, DOAJ, EBSCO Publishing’s Electronic Databases, Excerpta Medica / EMBASE, Genamics JournalSeek, Google Scholar, Health & Wellness Research Center, Health Reference Center Academic, Hinari, Index Copernicus, Indian Science Abstracts, Journal Citation Reports, National Science Library, OpenJGate, PrimoCentral, ProQuest, PubMed, Pubmed Central, Science Citation Index Expanded, Scimago Journal Ranking, SCOLOAR, SCOPUS, SIIC databases, Summon by Serial Solutions, Ulrich’s International Periodical Directory and Web of Science. Pharmacogn. Mag. ORIGINAL ARTICLE A multifaceted peer reviewed journal in the ield of Pharmacognosy and Natural Products www.phcog.com | www.phcog.net Toxicological Evaluation of Emblica oicinalis Fruit Extract and its Anti-inlammatory and Free Radical Scavenging Properties Sushil Kumar Middha, Arvind Kumar Goyal1, Prakash Lokesh2, Varsha Yardi5, Lavanya Mojamdar, Deepthi Sudhir Keni3, Dinesh Babu4, Talambedu Usha5 Departments of Biotechnology and 5Biochemistry, Maharani Lakshmi Ammanni College for Women, Malleswarm, 2Department of Biochemistry, CPGS, Jain College, Department of Biotechnology, Sapthagiri College of Engineering, Bengaluru, Karnataka, 1Department of Biotechnology, Bamboo Technology, Bodoland University, Kokrajhar, BTAD, Assam, 4Heymans Institute of Pharmacology, Ghent University, Belgium 3 ABSTRACT Background: Emblica oficinalis (Euphorbiaceae), popularly known as Indian gooseberry or “Amla” in India, is used in Ayurveda as “rejuvenating herb” since ancient times. Objective: This study was carried out to estimate toxicity, anti‑inlammatory, and antioxidative activities of the methanolic extract of Emblica oficinalis fruit (MEO) in an animal model. Materials and Methods: Antioxidative property of MEO was assessed by in vitro assays such as phosphomolybdenum assay (total antioxidant capacity), free radical scavenging assays 1,1‑diphenyl‑2‑picrylhydrazyl and 2,2’‑azino‑bis and 3‑ethylbenzthiazoline‑6‑sulphonic acid (DPPH and ABTS method) and lipid peroxidation assay (LPO). The anti‑inlammatory property was evaluated by carrageenan‑induced acute inlammation in rats by measuring rat paw volume at different time intervals and toxicological analysis using mice. Results and Discussion: High performance liquid chromatography studies revealed the presence of gallic acid (2.10%), mucic acid (4.90%), ellagic acid (2.10%), quercetin (28.00%), rutin (3.89%), and β‑glucogallin (1.46%). MEO showed highest antioxidant activities by using DPPH (17.33–89.00%), ABTS (23.03–94.16%), nitric oxide scavenging activity (12.94–70.16%), LPO (56.54%), and phosphomolybdenum assay (142 ± 6.09 µg/ml). The LD50 was found to be approximately 1125 mg/kg (p.o). High dose of MEO showed signiicant reduction (72.71%) in the inlammation after 4 h of treatment, which was comparable to diclofenac (10 mg/kg) (61.57%) treated group. Signiicant reduction (P < 0.05) in the inlammatory cytokine (interleukin‑1β and tumor necrosis factor‑α) markers were also observed (57.25% and 35.41%, respectively) in serum of MEO treated animals as compared to control. Conclusion: Taken together, phenolic compounds of MEO may serve as a potential herbal drug for amelioration of acute inlammation due to their modulatory action on free radicals. Key words: 1‑diphenyl‑2‑picrylhydrazyl, acute inlammation, amla, antioxidant, Emblica oficinalis, high performance liquid chromatography SUMMARY • The methanolic extract of Emblica oficinalis fruit (MEO) has potent antioxidant activity as assessed by DPPH, ABTS and LPO assays INTRODUCTION here is an escalating demand for herbal drug therapy, many plant species are being explored and are used in various human cultures around the world for their medicinal properties.[1] hese natural drugs help in maintaining the homeostasis. When cells are under stress, they produce free radicals like reactive oxygen species (ROS) and reactive nitrogen species (RNS) that damage the cells in many ways such as lipid peroxidation (LPO), protein denaturation, and DNA damage.[2] Nitric oxide (NO) is an important mediator of many physiological functions and its role in the inlammatory process is gaining recognition.[3] Natural antioxidants have an important role in protecting the cells against damage by ROS/RNS, which can lead to several alictions such as inlammation[4,5] and diabetes.[6] Inlammation is one of the irst responses to cell and tissue damage inlicted by diverse factors such as infections and chemicals and physical injuries. Inlammation is caused by the release of various inlammatory mediators such as cytokines ([interleukin [IL]‑1β, IL‑6), tumor necrosis factor alpha (TNF‑α), prostaglandins, bradykinin, histamine, growth factors, neurogenic factors, © 2015 Pharmacognosy Magazine | Published by Wolters Kluwer - Medknow • MEO has potent anti‑inlammatory activity in carrageenan induced paw edema model • The phenolic compounds of MEO might be a potential herbal drug for amelioration of acute inlammation. Abbreviations used: ROS, reactive oxygen species; RNS, reactive nitrogen species, LPO , lipid peroxidation, NO, nitric oxide, IL, interleukin; TNF α tumor necrosis factor alpha; NSAIDs, nonsteroidal anti inlammatory drugs; AA, ascorbic acid; MEO, methanolic extract of Emblica oficinalis fruit; ABTS+; 2,2’ azino bis 3 ethylbenzthiazoline 6 sulphonic acid; DPPH, 1,1 diphenyl 2 picrylhydrazyl; HPLC, high performance liquid chromatography; MDA, malondialdehyde; DMSO, dimethyl sulphoxide; Access this article online ELISA, enzyme linked immunosorbent assay. Website: www.phcog.com Correspondence: Quick Response Code: Dr. Talambedu Usha, Maharani Lakshmi Ammanni College for Women, Malleswarm, Bengaluru, Karnataka, India. E‑mail: ushatalambedu@gmail.com DOI : 10.4103/0973‑1296.168982 and followed by central sensitization and hypersensitivity.[7] During such conditions, nonsteroidal anti‑inlammatory drugs (NSAIDs) are used to alleviate pain and reduce inlammation.[8] However, long‑term use of these NSAIDs may lead to certain types of ulcers and other disorders,[9] and hence there is a prompt need to search for an alternative to these synthetic drugs where herbal medicines can be suitable candidates. This is an open access article distributed under the terms of the Creative Commons Attribution‑NonCommercial‑ShareAlike 3.0 License, which allows others to remix, tweak, and build upon the work non‑commercially, as long as the author is credited and the new creations are licensed under the identical terms. For reprints contact: reprints@medknow.com Cite this article as: Middha SK, Goyal AK, Lokesh P, Yardi V, Mojamdar L, Keni DS, et al. Toxicological Evaluation of Emblica oficinalis Fruit Extract and its Antiinlammatory and Free Radical Scavenging Properties. Phcog Mag 2015;11:427-33. S427 SUSHIL KUMAR MIDDHA, et al.: Anti-inlammatory properties of Emblica oicinalis fruit extract Emblica oicinalis Gaertn., syn. Phyllanthus emblica L., popularly known as Indian gooseberry, is a small to medium-sized deciduous tree, native to India and the Middle East, belonging to the family Euphorbiaceae. It is commonly known as “Amla” in India and is used in Ayurveda as “rejuvenating herb” since ancient times.[10] E. oicinalis extract contains several antioxidants such as emblicanin A and B, gallic acid, ellagic acid, ascorbic acid (AA) (Vitamin C), which are used to treat several medical conditions.[11] It also possesses several attributes like antipyretic, analgesic, antimicrobial, antifungal, antitussive, chemopreventive, immunostimulatory, hepatoprotective, cardioprotective, radioprotective, and potential to increase hemoglobin.[12] Despite its wide‑ranging medicinal uses, there are very few scientiic evidence showing correlation between in vivo anti‑inlammatory efects and antioxidant properties for this wonder berry. Hence, the present report emphasizes on in vivo anti‑inlammatory efectiveness of methanolic extract of Emblica oicinalis fruit (MEO) using carrageenan‑induced rat paw oedema model and a wide spectrum of in vitro antioxidant studies which includes models like 2,2’‑azino‑bis 3‑ethylbenzthiazoline‑6‑sulphonic acid (ABTS+), 1,1‑diphenyl‑2‑picrylhydrazyl (DPPH), molybdenum reduction method, NO determination, and LPO, along with quantitative analysis of potential antioxidant compounds of MEO by high performance liquid chromatography (HPLC). MATERIALS AND METHODS Chemicals and reagents Acetic anhydride, ammonium hydroxide, AA, butylated hydroxytoluene, 2‑deoxy‑2‑ribose, DPPH, ferric chloride, gallic acid, magnesium ribbon, quercetin, sodium nitrite, and trichloroacetic acid were obtained from HiMedia Laboratories, Pvt., Ltd., Mumbai, India. hiobarbituric acid was obtained from Loba Chemical, Mumbai, India. Ammonium persulphate, benzene, chloroform, di‑potassium hydrogen phosphate, ethanol, ethylenediaminetetraacetic acid, glacial acetic acid, hydrogen peroxide, methanol, 5% o‑phosphoric acid, 0.01% naphthylethylene diamine, potassium di‑hydrogen phosphate, potassium ferricyanide, potassium hydroxide, sodium hydroxide, and sulfanilamide were obtained from Rankem, Mumbai, India. ABTS and gallic acid (3,4,5‑trihydroxy benzoic acid) were obtained from Sigma, USA. Conc. HCl, conc. H2SO4, Fehling’s solution, Mayer’s reagents, and sodium carbonate were procured from Merck, Mumbai, India. Folin‑Ciocalteu reagent was from Sisco Research Laboratory, Mumbai, India. Aluminium chloride was obtained from SD Fine Chemicals Limited, Mumbai, India. All chemicals and solvents were of analytical grade. Preparation of plant material Methanolic extract was prepared using 500 g of coarse dried fruit powder, which was extracted in Soxhlet apparatus with 2 l of methanol for 24 h, in a three cycle process. hen, the mixture was iltered, concentrated, and lyophilized (LyoQuest, Telstar, Spain).[13] he MEO was dissolved in double‑distilled water in desired concentrations before analysis. In vitro assays 3‑ethylbenzthiazoline‑6‑sulphonic acid radical scavenging assay ABTS radical cations were produced by mixing ABTS and MEO and incubating the mixture at room temperature for 16 h in dark condition. his was followed by addition of 20 µl of 10 mM phosphate bufer saline (pH 7.4), test solutions of various concentrations, and 230 µl of ABTS radical solution (0.238 mM).[14] he absorbance was measured immediately at 734 nm spectrophotometer (Elico, SL 210). A control reaction was carried out without the test sample. Linear graph of concentration against percentage inhibition was constructed, and IC50 value was calculated. S428 1,1‑diphenyl‑2‑picrylhydrazyl radical scavenging assay Diferent concentrations of test solution and 50:l of DPPH (0.659 mM) solution was incubated at 25°C for 20 min, ater which the absorbance was read at 510 nm using spectrophotometer (Elico, SL 210).[15] A control reaction was carried out without the test sample. Linear graph of concentration against percentage inhibition was constructed, and IC50 value was calculated. he percentage inhibition was calculated according to the following equation: % inhibition = (A0 − At)/A0 × 100 where A0 was the absorbance of the control (blank, without extract), and At was the absorbance in the presence of the extract. Determination of total antioxidant capacity by phosphomolybdenum assay he total antioxidant capacity of MEO was assessed by phosphomolybdenum method.[16] To 0.3 mL of extract solution (1 mg/mL), 3 mL reagent solution (28 mM sodium phosphate, 6 M sulfuric acid and 4 mM ammonium molybdate) was added and incubated at 95°C for 90 min. hen, the absorbance of the solution was measured at 695 nm against blank spectrophotometer (Elico, SL 210). he antioxidant capacity of the extract was evaluated as AA equivalents. Lipid peroxidation assay Malondialdehyde (MDA), a marker of LPO was estimated by the method of Okhawa et al.[17] and as modiied by Middha et al.[18] using 1,1,3,3‑tetramethoxy propane as the standard, measured at 532 nm and expressed in nM/mg protein. Quantitative analysis of antioxidant compounds High‑performance liquid chromatography analysis HPLC (Waters Corporation, Singapore) protocol was followed as per Middha et al.[19] Active constituent in the MEO extract was dissolved in a mixture of methanol and water (6:4 v/v), and identiied by comparison of the retention time in chromatogram with standard Vitamin C, gallic acid, rutin, quercetin, and ellagic acid (Sigma Chemical Co., St Louis, USA). Data were analyzed using Empower sotware (Waters Corporation, Singapore). In vivo assay Animals Male Wistar albino rats (140–160 g) and Swiss albino mice (25–30 g) were ordered from Indian Institute of Sciences (Bangalore, India), housed in groups of 4 and given 7 days to acclimate to the housing facility in polypropylene, polycarbonate, and stainless steel cages. Rats were randomly selected and sheltered under standard laboratory conditions of light and dark cycles of 7:00 am to 7:00 pm, temperature of 25°C ± 2°C, lighting of 350 lux, and 68% ± 1% relative humidity and given access to rat maintenance food (Lipton India Ltd., Bangalore, India) and tap water ad libitum. Cages (size 421 mm × 290 mm × 190 mm with a gap of 7 mm between wires) and corn cob bedding were changed every alternate day to ensure that animals are clean and dry as per the National Institutes of Health guidelines.[20] During housing, animal’s health status was monitored twice daily, and no adverse events were detected. he study protocol was approved by Maharani Lakshmi Ammanni College Ethical Committee, clearance from Ethical Committee (1368/ac/10/CPCSEA), Bangalore, India. Acute toxicity test Swiss albino mice (25–30 g) of both sexes were divided into six groups of 10 each. Animals were housed 5 per cage and were starved overnight but were given water preceding the experiment. MEO was administered to each group at diferent dose levels of 0.5, 1.0, 1.5, 2.0, Pharmacognosy Magazine, Oct-Dec 2015, Vol 11, Issue 44 (Supplement 3) SUSHIL KUMAR MIDDHA, et al.: Anti-inlammatory properties of Emblica oicinalis fruit extract 2.5, and 3.0 g/kg BW/ml. he mice were kept under observation for 24 h, mortality was recorded, and LD50 (median lethal dose) was determined as per Middha et al.[18] RESULTS Yield of the extract he total yield of the extract was found to be 9.8%. Anti‑inlammatory activity The carrageenan‑induced rat paw edema test[21] was used as an experimental model for testing the anti‑inflammatory activity of MEO. The rats were divided into five groups of six rats each as shown in Table 1. The investigated MEO, dissolved in dimethyl sulphoxide (DMSO) were administered orally in doses of 200 mg/kg and 400 mg/kg; these doses were selected based on an initial toxicity screening for up to 24 h. Diclofenac (10 mg/kg) (Sigma Chemical Co., St. Louis, USA) dissolved in DMSO, was used as a reference drug. The control animals were given the vehicle (1 ml/kg p.o. DMSO). 1 h after the oral administration of the extracts or diclofenac, carrageenan‑saline solution (0.5% w/v) and saline were injected in a volume of 0.1 ml into the plantar surface of the right and left hind paw, respectively. Left paw served as the control (noninflamed) paw. The animals were observed for 0th h, 1st h, 2nd h, and 4th h; the paw volume was measured using plethysmometer (PanLab, Barcelona, Spain).[21] The anti‑inflammatory effect was calculated using the equation below: Anti‑inlammatory efect (%) = k − e/k × 100 where k is the diference in the paw weight in the control group and e is the diference in the paw weight in the treatment group. Finally, ater drug treatments, rats were euthanized using 0.3 ml/100 g i.p ketamine (300 mg/kg) +0.15 ml/100 g xylazine (30 mg/kg) as recommended by Committee for the Purpose of Control and Supervision of Experiments on Animals. Ater euthanization, the carcasses were placed in a non‑PVC containing, sealable, and transparent plastic bag and were sent to incinerator. Blood samples were collected by heart puncture method, and serum was separated for biochemical estimations. Measurement of interleukin‑1β and tumor necrosis factor‑α levels he serum concentration of IL‑1β was measured using a commercial enzyme‑linked immunosorbent assay (ELISA) kit method (Biosource, San Diego, California)[22] and TNF‑α was measured with another ELISA kit (Endogen, Woburn, MA, USA).[23] Statistical analysis In vitro activity 3‑ethylbenzthiazoline‑6‑sulphonic acid radical scavenging assay A dose‑dependent inhibition of scavenging activity was displayed by MEO to ABTS radical [Figure 1]. he percentage of ABTS scavenging by MEO was found to be at 23.03% and 94.16% at 10 µg/ml and 100 µg/ml, respectively. 1‑diphenyl‑2‑picrylhydrazyl radical scavenging assay MEO showed radical scavenging activity as measured by DPPH, across concentrations ranging from 0.1 to 1.0 µg/ml [Figure 2]. he values for maximum % inhibition at 0.1–1 mg/ml ranged from 7.33% to 83.33% in MEO, whereas that of standard (AA) was found to be 90%. he high DPPH scavenging activities of the methanolic extract have already been reported by other researchers.[24] IC50 of MEO was found to be 0.628 µg/ml. Determination of total antioxidant capacity by phosphomolybdenum assay he principle includes the reduction of Mo (VI) to Mo (V) by the extract that contains antioxidant compounds. In this study, MEO was more efective in reduction of Mo (VI) to Mo (V) (142.0 ± 6.09 µg/ml). he reduction of Mo (VI) to Mo (V) by administration of reference chemical, AA (IC50 81.3 ± 4.12 µg/ml), suggested the presence of efective antioxidants in MEO. Lipid peroxidation assay Decrease in the MDA levels is a good indication of an efective antioxidant. here was a signiicant dose‑dependent reduction in MDA levels by MEO. Maximum percentage reduction in MDA levels of 56.54% was observed at 300 µg/ml concentration of MEO. High performance liquid chromatography analysis HPLC method has been reported to be simple, easy to use, and efective enough for the identiication and quantiication of major phenolic compounds found in aromatic plants,[19,25] despite the fact that the separation of procyanidins is not satisfactory with these phases.[25] To deduce a better separation of phenolic acids All the measurements were repeated six times and expressed as a mean ± standard error of means and Statistically analyzed by two way analysis of variance followed by Tukey’s multiple ranges using GraphPad prism (GraphPad, San Diego, CA, USA) Table 1: Paw volume variation due to anti-inlammatory efects of methanolic extract of Emblica oicinalis fruit Experimental animals Dosages mg/kg Percentage increase 1st h 2nd h 4th h NL 2.60±0.09 2.90±0.007 2.20±0.019 IC No treatment 48.13±4.56 80.11±9.01a 92.84±10.136a IC + LMEO 200 42.67±1.93NS 68.89±3.50b 89.86±8.102a IC + HMEO 400 33.673±4.06NS 55.85±6.012c 72.71±7.242a IC + diclofenac 10 18.68±1.19a 50.68±0.012c 61.57±3.19b Diferent superscripts denote signiicant group at P<0.05 versus inlammatory group (IC). NSNot signiicant. IC + LMEO: Control rats treated with 200 mg/ml; IC + HMEO: Control rats treated with 400 mg/ml; IC + diclofenac: Control rats treated with diclofenac; NL: Normal; IC: Carrageenan injected control; SEM: Standard error of mean Pharmacognosy Magazine, Oct-Dec 2015, Vol 11, Issue 44 (Supplement 3) Figure 1: ABTS+ radical scavenging activity of methanolic extract of Emblica oicinalis fruit compared to standard ascorbic acid S429 SUSHIL KUMAR MIDDHA, et al.: Anti-inlammatory properties of Emblica oicinalis fruit extract and lavonoids in aromatic plants, we have used Spherisorb C18 stationary phase, which provided a quite ine outcome. Ater extraction and acid hydrolysis, the content of phenolic substances was determined. Quantiication was done via calibration with standards. he amount of phenolic acids such as quercetin (28.00 mg/100 g), rutin (3.89 mg/100 g), β‑glucogallin (0.146 mg/ml), gallic acid (2.10 mg/100 g), and mucic acid (4.90 mg/100 g) were detected in the analyzed samples [Figure 3]. Quercetin has been reported to be present in a large number of herbals.[19,26] Rutin in some cases can be hydrolyzed to quercetin (aglycone). his could have happened in our sample of interest. hus, hydrolysis was needed for the identiication and quantitative determination of phenolic acids. Time of harvest and storing conditions might have an efect on the variations in the phenolic compounds. Assessment of pharmacological activity (in vivo) Acute toxicity In vivo median lethal dose or LD50 of the extract was 1125 mg/kg BW. Other parameters such as body weight and survival rate showed no side efects to MEO (data not shown). Anti‑inlammatory activity Carrageenan‑induced paw edema: MEO signiicantly (P < 0.01) exhibited anti‑inlammatory activity in a dose‑dependent manner at 4 h. he reduction in carrageenan‑induced paw edema by high dosage of MEO (HMEO) ater 4 h was 72.71% while edema reduction by the standard drug, diclofenac (10 mg/kg) was 61.57% [Table 1]. MEO produced a dose‑dependent inhibition of paw edema, which was comparable to the efect of known anti‑inlammatory drug, diclofenac. Efect of methanolic extract of Emblica oicinalis on diferent cytokine levels Cytokines are small glycoproteins counteracted in response to an antigen and initially deined as a mediator for regulating the innate and adaptive immune reactions. At the time of inlammation, IL‑1β and TNF‑α are the two major cytokines, which play a major role in leading mechanisms.[7] he levels of these cytokines should be suppressed to reduce the severity of the inlammatory reaction. Table 2 indicates that inlammatory reaction induced by carrageenan signiicantly increased the concentration of IL‑1β and TNF‑α (P < 0.05). MEO treatment decreased the level of these cytokines signiicantly as compared to the inlammation‑induced group. DISCUSSION Figure 2: DPPH scavenging activity of methanolic extract of Emblica oicinalis fruit compared to standard ascorbic acid Quite a lot of present day diseases are due to the shit in the balance of pro‑oxidant and antioxidant homeostasis in the body.[13,27] Inlammation is the response to injury of cells and body tissues through diferent factors such as infections and chemicals and mechanical injuries.[22,28] he pathogenesis of inlammation is brought about by free radicals.[22,29,30] Plants have many compounds which show diferent bioactivities, including antioxidant and anti‑inlammatory, which are experimented on because of their numerous mechanisms of actions with non‑toxic nature.[19] Antioxidants interfere with the oxidative processes by scavenging free radicals and chelating free catalytic metals.[31] here are numerous NSAIDs existing in the market, but these drugs have been shown to have certain side efects that may cause cardiovascular and gastrointestinal disorders; therefore, it is important to endorse the drugs from natural sources.[32] Figure 3: High performance liquid chromatography chromatogram of ascorbic acid (1), quercetin (2), rutin (3), gallic acid (4), mucic acid (5), and beta-glucogallin (6) in methanolic extract of Emblica oicinalis fruit S430 Pharmacognosy Magazine, Oct-Dec 2015, Vol 11, Issue 44 (Supplement 3) SUSHIL KUMAR MIDDHA, et al.: Anti-inlammatory properties of Emblica oicinalis fruit extract Table 2: Cytokines levels IL-1β and TNF-α of experimental animals (n=6) IL‑1β (pg/mg protein) TNF‑α (ng/µg protein) NL 3.2±0.02 0.2±0.001 IC 12.4±0.12 0.48±0.002 IC + LMEO (200 mg/kg) 7.8±0.04* 0.39±0.005* IC + HMEO (400 mg/kg) 5.3±0.07** 0.31±0.006** IC + diclofenac 4.9±0.04*** 0.28±0.008** Values are in means±SEM. *P<0.05 versus inlammatory group; **P<0.01 versus inlammatory group; ***P<0.001 versus inlammatory group. IC + LMEO: Control rats treated with 200 mg/ml; IC + HMEO: Control rats treated with 400 mg/ml; IC + diclofenac: Control rats treated with diclofenac; NL: Normal; IC: Carrageenan injected control. SEM: Standard error of mean; IL‑1β: Interleukin‑1β; TNF‑α: Tumor necrosis factor‑α Emblica has been used for the treatment of arthritis and other inlammatory diseases.[32] A wide variety of in vitro methods such as DPPH and ABTS are used to assess radical scavenging ability of certain agents from natural and synthetic sources. DPPH is a stable free radical, and its activity is based on its ability to decolorize from deep purple color at 517 nm to light yellow in the presence of antioxidants forming a stable DPPH molecule.[33] MEO showed an efective radical scavenging ability by this assay. ABTS is a blue colored chromophore and upon addition of MEO, it is reduced and this reduction process is concentration dependent. E. oicinalis is a rich source of Vitamin C, alkaloids, phenolics, lavonoids, and tannins.[12] In agreement to this, our study revealed that the MEO has a high level of radical scavenging capacity as evidenced in both DPPH and ABTS assays. his might be because of more phenolic compounds in MEO, which help in scavenging of radicals in the two studied assays and hence attribute to their higher antioxidant activity.[13,33] he phosphomolybdate results further conirmed the potent antioxidant activity of MEO, which increased in a concentration‑dependent manner. In vivo toxicological estimation was performed to evaluate the extent to which a drug can cause deleterious efects to the cells; these tests are essential and widely accepted to know the optimum drug concentration to be administered. LD50 of MEO was measured to check lethal toxicity and was found to be 1125 mg/kg BW. Treatment with MEO at a dose of 200mg/kg and 400mg/kg to their respective group of mice for 1 month did not show any toxic side efect. he selection of the 2 doses 200 mg/kg and 400 mg/kg was based on an initial toxicity study where until 1 month ater administration of these doses of the extract, the animals were healthy without any visual signs or symptoms of illness (data not shown). Previously, Golechha et al., 2014[34] showed that a 300–500 mg/kg dose of Emblica has not shown any adverse efect. Carrageenan induced edema of rat paw is used widely as a working model of acute inlammation in search for new anti inlammatory drugs.[35] During the irst 2 h (Phase I), liberation of histamine and serotonin begins in rat paw ater carrageenan injection, whereas in Phase II release of kinin, prostaglandin, bradykinin, leukotrienes, and polymorphonuclear cells in the damaged tissue in surroundings cells also increases depending upon the complement system for their activation.[34] Results of standard drug diclofenac sodium, normal saline, and two doses of test extracts (LMEO and HMEO) were compared. Both the tested doses of MEO have shown a signiicant reduction in paw volume as compared to control group; the eiciency of MEO is very much comparable to that of the standard drug (P < 0.05). Our in vivo results were strongly in accordance to previous studies.[36] In case of infection, inlammation, and immune activation, IL‑1β and TNF‑α play a major role. Sometimes, TNF‑α can also stimulate the release of IL‑1β.[4] he levels of IL‑1β and TNF‑α of experimental animals showed a signiicant decrease ater administration of MEO as compared to the Pharmacognosy Magazine, Oct-Dec 2015, Vol 11, Issue 44 (Supplement 3) saline‑treated animal group. Rats treated with high dosage (400 mg/ml) showed more potent efect than lower dosage (200 mg/ml) which is very much comparable to the standard drug. he role of phenolic compounds in inlammatory conditions is well established.[37,38] hey also inhibit polymorphonuclear lipoxygenase, an enzyme involved in inlammatory conditions.[37] In this context, E. oicinalis has caught the attention of the researchers as it is a potential source of phenolic compounds and lavonoids.[36] hese efects may be due to high levels of quercetin, rutin, and other phenolic compounds found in the MEO.[23,28] CONCLUSIONS In summary, the results of this study suggest that MEO not only is a storehouse of natural antioxidant but also possesses strong anti‑inlammatory activity which might be helpful in preventing or slowing the progress of various oxidative stress‑related diseases. he anti‑inlammatory activity of MEO may probably be due to the presence of several bioactive anti‑inlammatory principals as elucidated by HPLC analysis. However, it needs isolation, structural elucidation, and screening of any of the various active principle (s) to point out the real activity of the drug. Even, an extensive clinical study needs to be hypothesized to ascertain the eicacy and safety of this agent to illustrate an improvement over currently available treatment. Acknowledgments his work was partially supported by the grants from BT‑Finishing School, Govt. of Karnataka and Maharani Lakshmi Ammanni College for Women, Bangalore. Financial support and sponsorship Nil. Conlicts of interest here are no conlicts of interest. REFERENCES 1. Middha SK, Mittal Y, Ushal T, Kumar D, Srinivasan R, Vashisth L, et al. Phyto‑mellitus: A phyto‑chemical database for diabetes. Bioinformation 2009;4:78‑9. 2. Goyal AK, Basistha BC, Sen A, Middha SK. Antioxidant proiling of Hippophae salicifolia growing in sacred forests of Sikkim, India. Funct Plant Biol 2011;38:697‑702. 3. 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Anti‑inlammatory, anticholinesterase, and antioxidant radical‑scavenging activity of phenol components of Emblica oficinalis extract by using an potential of scopoletin isolated from Canarium patentinervium Miq. (Burseraceae Kunth). HPTLC‑DPPH* method. J Sep Sci 2007;30:1250‑4. Evid Based Complement Alternat Med 2013;2013:734824. S432 Pharmacognosy Magazine, Oct-Dec 2015, Vol 11, Issue 44 (Supplement 3) SUSHIL KUMAR MIDDHA, et al.: Anti-inlammatory properties of Emblica oicinalis fruit extract ABOUT AUTHORS Sushil Kumar Middha: Currently working as a Lecturer/Assistant Professor at the department of Biotechnology, Maharani Lakshmi Ammanni College For Women, Bangalore, India and having expertise in bioinformatics tools, in silico drug discovery methods, pre clinical studies and antioxidants. Arvind Kumar Goyal: Currently working as a guest instructor at the Bamboo technology, department of Biotechnology and having expertise in Bamboo tissue culture, biomarkers (RAPD, AFLP) and Phytochemistry. Sushil Kumar Middha Arvind Kumar Goyal Dinesh Babu: Currently working as a research scholar at the department of Gastro‑intestinal Neuropharmacology, Heymans Institute of Pharmacology, Faculty of Medicine and Health Sciences; Ghent University, Belgium and having expertise in vitro studies (cell lines method) and antioxidant assays. Prakash Lokesh: Currently working as a Lecturer/Assistant Professor at the Jain University and proicient in handling HPLC and having expertise in Phytochemistry and Proteomics. Varsha Yardi: Student in Biochemistry at the Maharani Lakshmi Ammanni College For Women, Bangalore, India. Prakash Lokesh Varsha Yardi Lavanya Mojamdar: Student in Biotechnology at the Maharani Lakshmi Ammanni College For Women, Bangalore, India. Deepthi Sudhir Keni: Student in Biotechnology at the Sapthagiri College of Engineering, Bengaluru, Karnataka, India. Talambedu Usha: Currently working as a Lecturer/Assistant Professor at the department of Biochemistry, Maharani Lakshmi Ammanni College For Women, Bangalore, India and having expertise in biochemical assays, bioinformatics tools and antioxidants. Talambedu Usha Dinesh Babu Lavanya Mojamdar Deepthi Sudhir Keni Pharmacognosy Magazine, Oct-Dec 2015, Vol 11, Issue 44 (Supplement 3) S433