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Journal of Pharmacy Research Vol.5 Issue 3.March 2012 Sumathy Haridass et al. / Journal of Pharmacy Research 2012,5(3),1403-1408 1403-1408 Research Article ISSN: 0974-6943 Available online through http://jprsolutions.info *Corresponding author. H.Sumathy Department of Biochemistry Bharathi Women’s College North Chennai, Tamil Nadu, India INTRODUCTION Relative antioxidant and cytotoxic activities of Ixora coccinea flower extracts Sumathy Haridass1*, Sathiya Sekar2, Ranju Vijayan2, Sangeetha Jayakumar1, Sadhana Thtmizharasu2, Vijayalakshmi Krishnamurthy1 and Saravana Babu Chidambaram2 1Department of Biochemistry, Bharathi Women’s College,North Chennai, Tamil Nadu, India 2Centre for Toxicology and Developmental Research, Sri Ramachandra University, Porur, Tamil Nadu, India Received on:10-12-2011; Revised on: 15-01-2011; Accepted on:12-02-2012 ABSTRACT Objective: To investigate the presence of various phytochemicals from the flowers extracts of Ixora coccinea belonging to the Rubiaceae family. Methods: Quantitative estimations were performed for tannin, phenol, flavonoids and terpenoids. In vitro antioxidant potential of the three extracts were performed using DPPH assay, Superoxide anion radical scavenging assay, LPO assay, hydrogen peroxide (H2O2), nitric oxide (NO) scavenging assays and MTT assay. Results: Preliminary phytochemical analysis revealed the presence of tannin, phenol in Methanol extract, tannin, phenol, flavonoid and terpenoids were found to be present in ethyl acetate extract and none of phytochemicals was seemed to be present in high amount in petroleum ether extract. Quantitative determination revealed the presence of high amount of Tannin (68.74±4.26 mg/g extract)and total phenol (216.78±8.49 mg/g extract) in Methanolic extract flavonoids (41.46±1.17mg/g extract) and terpenoids (207.47±8.76 mg/g extract) in ethyl acetate extract where as in petroleum ether none of these phytochemicals were found to be present in high amounts. Ethyl acetate extract was found behave high total antioxidant capacity (0.63 ± 0.04 gm extract which contains equivalent of Vit E). The EA extract was found to be potent in scavenging free radicals like DPPH (12.86±1.14 µg/10µl), LPO (121.72±7.17µg/ml), NO (48.57±3.85µg/0.5ml) and H2O2 (27.54±1.39). ME and EA were found to have equal potential in scavenging the other radicals like superoxide anion and hydroxyl radical. Cytotoxic potential was high in EA (42.28±1.63µg/µl). Conclusion: These results reveal that EA extract of I.coccinea has strong antioxidant potential compared with other extracts. Further study on the in vitro anticancer activity was extended to elucidate the molecular Mechanism of action of the EA extract. Key words:Ixora coccinea Ethyl acetate Flavonoids Terpenoids Total antioxidants Free radicals Cytotoxicity Plants show enormous versatility in synthesizing complex materials, which have no immediate obvious growth or Metabolic functions. These complex materials are referred to as secondary Metabolites. Secondary Metabolites present in plants are biologically active and these bioactive compounds are believed to be effective in combating or preventing disease due to their antioxidant effect which is due to their capability to quench lipid peroxidation, prevent DNA oxidative damage and scavenge reactive oxygen species. Oxidative modification of macromolecules like DNA, proteins, lipids and small cellular molecules by free radicals plays a major role in numerous diseases and age related degenerative conditions. Free radicals play an important role, in both health and disease, and have been implicated in manifold human disease processes. The antioxidant and antimicrobial properties of various plants have been reported by several studies. Abundant natural antioxidants have been reported in fruits and vegetables[2,3,4], oilseeds, herbs, tea. Antioxidant defence systems can reduce side effects induced by ROS in living cells. Synthetic antioxidants, such as butylated hydroxy anisole (BHA) and butylated hydroxy toluene (BHT) are widely used in the food and cosmetic industry. It has been reported that administration of high dose of BHT was found to be hepatotoxic and induce lung damage[8,9] and long term administration of BHT is capable to induce oxidative and metabolic alterations in heart similarly to some pathological disorders. World health organization has recommended the evaluation of the plants effective in conditions were safe modern drugs are lacking. Recently, an intensive search for novel sources of antioxidants in the last years has been extensively studied for their antioxidant activity. Ixora coccinea (Rubiaceae), a small to medium sized hardy shurb cultivated for ornamental purpose were also used in traditional Indian medicine. Antimicrobial activity of I.coccinea leaves and flower extracts have been 1.MATERIALS AND METHODS 1.1.Chemicals and instruments DPPH (2,2-diphenyl-picrylhydrazyl) radical, PMS, NBT, NADH, sulphanilamide, sodium nitroprusside, napthyl ethyldiamine, deoxyribose, hydrogen peroxide, ferric chloride, trichloro acetic acid, thio barbutric acid, potassium dihydrogen phosphate and ferrous sulphate ascorbic acid were purchased from SRL chemicals India Ltd. DMEM, FBS, Penicillin and streptomycin were obtained from GIBCO, USA. All other chemicals and solvents used were of analytical grade. The absorbance measurements were recorded using the UV-Visible Thermo multiskan spectrophotometer. 1.2.HeLa cell lines and culture Medium HeLa cell line was procured from National Centre for Cell Sciences (NCCS), Pune, India. Cells were grown in DMEM Medium with 10% FBS, 100 U/ ml penicillin and 100 µg/ml streptomycin and Non essential amino acids and sodium pyruvate. The cells were grown in a humidified atmosphere in a CO2 incubator at 37°C with 5% carbon dioxide. reported[11,12]. Anti-inflammatory and antimitotic activities from leaf extracts have been reported. They have also been reported to have anti-inflammatory activity comparable to indomethacin. Flowers were also reported to possess cytotoxic and antitumour activity in mice injected with Dalton’s lymphoma ascetic (DLA) cells. Flowers extracts were reported to contain triterpenoid, ursolic acid. The flowers afforded two new cycloartenol esters, lupeol fatty ester, lupeol, oleanolic acid and sitosterol. Flowers showed protective effects against cyclophosphamide and cisplastin induced systemic toxicity[15,16]. Wound healing properties of alcoholic extract of flower was also reported and it was shown to significantly increase the enzymatic profiles of Wistar rats. Journal of Pharmacy Research Vol.5 Issue 3.March 2012 Sumathy Haridass et al. / Journal of Pharmacy Research 2012,5(3),1403-1408 1403-1408 1.3.Plant material and extraction Ixora coccinea were collected from local herbal drug vendor and were authenticated by Dr.Amarjothi, Department of Botany, Presidency College, Chennai, India. Flowers were washed thoroughly with water to remove the earthy matters and freed from debris. Raw flowers were shade dried, powered (80% coarse: 20% fine) and subjected to successive (petroleum ether, ethyl acetate and methanol) extraction by hot continuous percolation Method using Soxhlet’s apparatus. Extracts were concentrated under vacuum, in rotary evaporator, dried and stored in vacuum desiccators till use. radical scavenging activity of test material and was performed according to Koleva. DPPH was measured by adding 190µl of DPPH (3mg of was dissolved in 50ml ethanol) to 10µl of different concentrations (1.5-1000µg) of the extracts and incubated at room temperature for 30 min. Absorbance was read at 517 nm. The antioxidant activity was calculated as inhibition (%) of DPPH radical formation. % DPPH = (Acontrol - Asample) /Acontrol X 100 1.12.Superoxide anion radical scavenging activity Superoxide anion radical scavenging activity was assayed using the method of Kakkar by taking 100µl of different concentrations of extracts (1.5- 1000µg), 250µl of pyrophosphate buffer (0.025M, pH8.3), 25µl of PMS and 75µl of NBT. Reaction was started by addition of 7µl of NADH, 250µl of acetic acid and 2ml of butanol. After incubation of the reaction mixture at 37 ºC for 10 sec the tubes were centrifuged at 3500rpm for 10min. 2ml of n- butanol alone served as blank. The colour intensity was read at 560nm. Percentage inhibition was calculated by comparing the results of control and test samples. % SOD = (Acontrol - Asample) /Acontrol X 100 1.13.Determination of inhibition of Lipid peroxidation Lipid peroxidation was evaluated by measuring the TBARS content according to the TBA test described by Ohkawa with slight modifications. To different concentration of the extracts ((1.5-1000µg)) 1ml of 10% homogenate, 0.1ml of ferrous sulphate, 0.1ml of ascorbic acid, 0.1ml of potassium dihydrogen phosphate and 2.7mlof water was added and incubated at 37C for 1hr. Then, 1ml of 5% TCA and 1mlof 0.375% of TBA was added and the tubes were boiled for 30mins. After cooling, the solution was centrifuged at 3000rpm for 10mins. The intensity of color is the measure of MDA concentration. Absorbance at 532nm was determined using UV/ VIS spectrophotometer against the blank. % LPO = (Acontrol - Asample) /Acontrol X 100 1.14.Nitrate / Nitrite radical scavenging activity Aqueous sodium nitroprusside at physiological pH spontaneously generates nitric oxide (NO), which interacts with oxygen to produce nitrite, which can be estimated by use of Greiss reagent. Nitrate/Nitrite was (NO2/NO3) was assayed by taking 0.5ml of extracts of various concentration (1.5-1000µg) followed by addition of 1.25ml of phosphate buffer, 1.25ml of sodium nitroprusside and incubated at room temperature for 2 and half hours. To the mixture, 1.25ml of Griess reagent (1g of sulphanilamide dissolved in small volume of water, 2 ml of orthophosphoric acid and 100mg of naphtyl ethyldiamine were added. Volume was made upto 100ml with distilled water and mixed well) was added. Intensity of colour developed was read 546 nm. % NO = (Acontrol - Asample) /Acontrol X 100 1.15.Hydroxy (·HO) radical scavenging activity Among oxygen-centered radicals, the hydroxyl radical (·OH) is the most reactive and can damage numerous biomolecules. Hydroxy radical scavenging activity was assayed according to the method of Halliwel, by adding 1ml of extracts of various concentration (1.5-1000µg) to 360µl of deoxyribose and 100µl of EDTA, 10µl of ferric chloride, 100µl of hydrogen peroxide and 330µl of phosphate buffer (50mM, pH7.4). The mixture was preincubated for 30mins at 37ºC. Then, 0.1ml of ascorbic acid was added and left undisturbed for 10mins. The reaction was arrested by adding 1ml of 10% TCA and 1ml of 0.5% TBA was added and the tubes were boiled for 30mins, cooled and then centrifuged at 3500rpm and the colour intensity was read at 532nm. % OH = (Acontrol - Asample) /Acontrol X 100 1.16.H2O2 Radical scavenging activity Hydrogen peroxide radical scavenging activity was assayed by the method of Sinha with slight modification. Reaction was started by taking 100µl of different concentrations (1.5-1000µg) of extracts, 500µl of buffer and 400µl of 2mM hydrogen peroxide. The reaction mixture was incubated at 1.7.Determination of total flavonoid content Aluminium chloride colorimetric method was used for flavonoids determination. 0.5ml of the extracts was mixed with 0.1ml of 10% aluminium chloride, 0.1ml of 1M sodium acetate and the volume was made upto 5.3ml with distilled water and incubated at room temperature for 30min, the absorbance of the reaction mixture was measured at 420nm. Quercetin was used as standard. 1.8.Determination of Triterpenoids Terpenoid was estimated by the method of Ing–Luen et al., 2009. 0.2 ml of extract prepared in ethanol (mg/ml concentration) was evaporated by keeping it in boiling water bath and to the residue 0.3ml of Vannilin / glacial acetic acid (W/V), 1ml of perchloric acid was added and incubated at 60° C for 45min. Tubes were cooled in ice and to the mixture 5ml of glacial acetic acid was added and the color intensity was absorbed at 548nm. 1.9.Reducing power Reducing capacity of the vitamin c was evaluated by standard procedures. Briefly, 0.5 ml of extract was added to 0.1ml of DTC reagent (0.4g of thiourea, 0.05g of copper sulphate and 3g of DNPH) and incubated at 37°C for 3 hrs. To the mixture 0.75ml of 85% hydrochloric acid was added and incubated at room temperature for 30mins. Intensity of color developed was read at 520 nm using a spectrophotometer. 1.10.Total antioxidant capacity Total antioxidant capacity was assayed by adding 0.1ml of extract to 1 ml of vitamin E reagent (0.6M sulphuric acid, 0.028M sodium dihydrogen phosphate and 4mM ammonium molybdate). Then, mixture was incubated at 60°C for 30mins. Color developed was measured at 695nm using a spectrophotometer. 1.11.Antioxidant assay with 1, 1-diphenyl-2-picrylhydrazyl (DPPH) radical DPPH is a free radical, when dissolved in ethanol has a violet color which when reacts with reducing agent, the solution loses its color that depends upon the number of electrons taken up. Hence, the loss of color indicates 1.4.Phytochemical screening Qualitative determination of phytochemical constituents of the three extract were carried out by standard protocols[18,19,20]. 1.5. Determination of tannin content Tannin content was determined by the Method described by. To 0.5ml of extracts (mg/ml concentration), 0.5ml of Folin’s phenol (1:2 radtio) and 1% 5ml sodium carbonate was added and incubated for 5 mins at room temperature. 0.5ml of folin’s phenol and sodium carbonate alone served as blank. Intensity of blue coloration of the reaction mixture was read at 640nm. Results were expressed as milligrams of gallic acid equivalent per gram of dry weight (mg GAE/g dw). 1.6.Determination of total phenol content The amount of total phenolics in all the extracts was determined by employing Folin’s Ciocalteau reagent. Reaction of 0.5ml of each sample was mixed with 2.5ml of Folin’s phenol and 2ml of sodium carbonate and incubated for 30mins at room temperature. Intensity of color developed was read at 765nm. 2.5ml of Folin’s phenol, 2ml of sodium carbonate and the solvent system served as blank. Results were expressed as milligrams of gallic acid equivalent per gram of dry weight (mg GAE/g dw). Journal of Pharmacy Research Vol.5 Issue 3.March 2012 Sumathy Haridass et al. / Journal of Pharmacy Research 2012,5(3),1403-1408 1403-1408 room temperature for 5min. Then, to the reaction mixture 2ml of dichromate acetic acid reagent (5% Potassium dichromate and Glacial Acetic Acid in 1:3 v/v) and the decrease in color intensity was measured at 570nm. 2ml of dichromate acetic acid reagent alone served as blank whereas the reaction mixture without extracts served as control. % H2O2 = (Acontrol - Asample) /Acontrol X 100 1.17. Cytotoxicity assessment Cell growth inhibition was determined by the 3-(4, 5- diMeOHthylthiazol-2-yl)-2,5 diphenyl-tetrazolium bromide (MTT) quantitative assay capable of detecting viable cells. Cells were plated in 96 well microtiter (ELISA) plates at an initial density of 7000 cells/well. After incubation for 24 h at 37°C, cells were treated with different concentrations (ranging from 100µg-1pg) of various extracts of IC and incubated for 18 h. MTT solution was added to each well and further incubated for 4 h at 37°C, optical density was read with an ELISA reader at 550 nm with 670 nm as the reference range. 1.18.Statistical Analysis Triplicate of each sample were used for statistical analysis. Data were expressed as Mean±SD. The significance between the results was assessed using the Student’s t-test and significance was accepted for p-values
The medicinal values of Ixora coccinea Linn. (Raktaka) has been recorded since ancient times. It belongs to family Rubiaceae. The roots and leaves are used in treating various ailments. The flower too has therapeutic potentials. Although the flowers are used as medicine by traditional healers it is not known too many. The current study is carried out to provide scientific details in the identification and the authenticity of I. coccinea Linn. floral parts with the help of pharmacognostical standards.
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