Int. J. Pharm. Sci. Rev. Res., 39(2), July – August 2016; Article No. 17, Pages: 93-99 ISSN 0976 – 044X Research Article Anti-Inflammatory and Antioxidant Activities of Rhubarb Roots Extract Eman A. Ibrahim1, Doha H. Abou Baker2, Farouk K. El-Baz1* 1Plant Biochemistry Department, National Research Centre (NRC), 33 EL Bohouth St. (Former EL Tahrir st.), Dokki, Giza, Egypt. 2Medicinal and Aromatic Plant Department, National Research Centre (NRC), 33 El Bohouth St. (former El Tahrir st.), Dokki, Giza, Egypt. *Corresponding author’s E-mail: [email protected] Accepted on: 02-06-2016; Finalized on: 31-07-2016. ABSTRACT Rhubarb (Rheum rhaponticum L.) is considered one of the commonly used edible and medicinal plant. This work was carried out to investigate the anti-inflammatory and antioxidant activities of Rhubarb roots extract. Total tannin, total phenolic and total flavonoid contents of ethanolic and water extracts as well as phenolic and flavonoid compounds were also determined in Rhubarb roots. The highest content of total tannin, phenolic and flavonoid were found in ethanolic extract. The major phenolic acids were benzoic and ferulic acids followed by vanillic acid, while the major flavonoid was narengine. The results indicated that the highest antioxidant and anti-inflammatory activities were found in ethanolic extract and highly correlated with phenolic content. The results of the current work indicated that ethanolic extract has the potential to be used as a natural anti- inflammatory and antioxidant. Keywords: Rhubarb roots, anti-inflammatory, antioxidant. INTRODUCTION and shacked for 24 h at room temperature. The extraction was repeated twice and then filtered. The hubarb is a perennial plant belonging to resulting of different extracts was used for the Polygonaceae. It also, considers a common determination of total tannin, phenolic, flavonoid, as well vegetable known as sibiric Rhubarb.1 This herbis a R 2 as their anti-inflammatory and antioxidant activities. storehouse of a large number of important crude drugs. Phytochemical investigation on Rhubarb has proved Quantitative Analysis major bioactive ingredients arephenolic compounds in six Total Tannin Content skeletal type including anthraquinones (physcion, chrysophanol, emodin, aloe-emodin and rhein and their Total tannin content was determined using the Folin- glucosides), anthocyanins (cyanidin 3-rutinoside and Ciocalteu reagent assay according to Tambe and cyanidin 3-glucoside), flavonoids (catechin, quercetin 3-O- Bhambar.16 About 0.1 ml of Rhubarb roots extract or rhamnoside, quercetin3-O-galactoside, and quercetin 3- standard solutions of (tannic 20-120 mg/l) was added to O-rutinoside), stilbene (trans-rhapontigenin and 7.5 ml distilled water then 0.5 ml of Folin reagent and 1 desoxyrhapontigenin (cis-rhapontigenin, resveratrol and ml of 35% sodium carbonate solution were added. The piceatannol).3 volume was made up for 10 ml with distilled water and the absorbance was measured against blank at 725 nm by Rhubarb is well-known for its strong biological activities using spectrophotometer. such ascathartic, anticancer, hepatoprotective, anti- inflammatory, anti-diabetic, analgesic, antiplatelet, Total Phenolic Content antibacterial, anti-oxidative, and antimutagenic effects.4- Total phenolic in Rhubarb roots ethanolic and water 11 extracts were determined by using Folin-Ciocalteu’s Rhubarb roots are used as oriental laxative medicine and reagent.17 The reaction mixture was prepared from an antipsoriatic drug, also used against diarrhea, as well mixing 0.5 ml of Rhubarb ethanol extract, 2.5 ml of 10% as stomachic, antiemetic, hemorrhoids, measles, Folin-Ciocalteu’s reagent dissolved in water and 2.5 ml 12,13 smallpox and cholagogue. Rhubarb also showed the 7.5% NaHCO3. The mixtures were incubated in a protective effect against liver injury and fatty liver.14-15 thermostat at 45oC for 45 min. The absorbance was spectrophotometrically recorded at 760 nm. Standard The purpose of the current work was to investigate the series concentrations of Gallic acid were prepared in anti-inflammatory and antioxidant activities of Rhubarb ranges 2-12 µg/ml and treated as the samples. roots extract. Total Flavonoid Content MATERIALS AND METHODS Total flavonoid content of Rhubarb roots extract was Preparation of Plant Extracts determined by the aluminum chloride method using Rhubarb was purchased from local market in Egypt. The quercetin as a standard.18 0.2 ml of the extracts or Rhubarb roots were extracted, briefly, 10 g of the dried standard solutions (quercetin, 20–120 mg/l) was mixed powder were soaked with 100 ml water and 80% ethanol with 0.3 ml 5 % NaNO2. After 5 min, 0.3 mL of 10% AlCl3 International Journal of Pharmaceutical Sciences Review and Research International Journal of Pharmaceutical Sciences Review and Research Available online at www.globalresearchonline.net 93 Available online at www.globalresearchonline.net © Copyright protected. Unauthorised republication, reproduction, distribution, dissemination and copying of this document in whole or in part is strictly prohibited. Int. J. Pharm. Sci. Rev. Res., 39(2), July – August 2016; Article No. 17, Pages: 93-99 ISSN 0976 – 044X was added then incubated for 6 min and 2 mL of 1 mol/L solution. After incubation for 30 min in the dark, the NaOH was subsequently added. The solutions were mixed absorbance was measured at 517nm. well and the absorbance was measured against prepared The percentage of DPPH scavenging activity was reagent blank at 510 nm by using spectrophotometer. calculated using the following formula: Identification of Phenolic and Flavonoid Compounds [ ( )] The phenolic and flavonoid compounds of Rhubarb roots were extracted according to the method described by ABTS Free Radical Scavenging Assay Hakkinen19 and Mattila.20 The ABTS free radical scavenging activity assays were Ten grams sample were extracted using 10 ml of aqueous carried out according to Arnao.23 and 80% ethanol by homogenization for 2 min then Potassium persulfate (2.6 mM) was added to 7.4 mM of centrifuged at 25,000 g for 10 min. The supernatant was decanted into a round-bottom flask. The pellet was re- ABTS+ and kept for 12–16 h at room temperature in dark. suspended in aqueous and 80% ethanol, centrifuged, and The ABTS+ solution (1mL) was diluted with 60ml the supernatants were combined, evaporated by rotary at methanol to an absorbance of 1.1 ± 0.02 at 734 nm 40 °C until dryness and resolved in methanol-HPLC. before analysis. Extracts were filtrated through 0.20µm millipore ABTS+ solution (2.80 mL) was added to sample fractions membrane filter and set up to a known volume (10 ml). (0.150 mL, 50–150 µg/mL). Three milliliters were collected in a vial for subsequent HPLC separation. HPLC instrument (Hewlett Packard, The After incubation for 2h in the dark, the absorbance series 1050, country) equipped with column C18 hypersil was measured at 734nm. BDS with particle size 5μm. Injection volume was 75 µl Trolox was used as the positive control. The ABTS free carried out with autosamplling injector. The column radical-scavenging activity (%) was calculated using the temperature was maintained at 35°C. Gradient following equation: separation was carried out with methanol and acetonitrile as a mobile phase at flow rate 1.0 ml/min. [ ( )] Elutes were monitored using UV detector set at 280 nm for phenolic acid and 330 nm for flavonoid. Where, A0 was the absorbance of the control (without Chromatographic peaks were identified by comparing the sample), A1 was the absorbance in the presence of the retention times with the respective retention times of sample, and A2 was the absorbance without ABTS+). known standard reference material. Retention time and peak area were used to calculation of phenolic acid and Total Antioxidant Activity flavonoid compounds concentration by the data analysis Total antioxidant activity assay was carried out according of Hewlett Packard software. Phenolic acid and flavonoid to Prieto.24 compounds were expressed as µg/100g sample on dry weight basis. One ml of different extracts from Rhubarb roots ethanolic and water extracts (100 to 400 µg/mL) were mixed with 3 In-vitro Anti-inflammatory Activity ml of reagent solution (0.6 M sulfuric acid, 28mM sodium Anti-inflammatory activity of different extracts from phosphate, and 4 mM ammonium molybdate). Rhubarb roots extract was carried out using the method The tubes were capped and incubated at 95°C for 90 min. of Rahman.21 The different concentrations of Rhubarb roots extract or standard drug diclofenac sodium (50, After cooling, the absorbance was measured at 695 nm. 100, 150,200 ug/ml) were mixed with 0.45ml bovine Standard series concentrations of ascorbic acid were albumin serum. The mixtures were incubated at 37°C for treated as the sample. 20 min and then heated to 57°C for 3 min, after cooling Statistical Analysis 2.5ml phosphate buffer PH 6.4 were added to the samples. The absorbance was measured at 255nm using All results are expressed as mean value of three replicate. UV visible spectrophotometer. Data were statistically analyzed through analysis of Antioxidant Activity variance (Anova) and Duncans test at P≥0.01 using CoStat Statistics Software. DPPH Free Radical Scavenging Assay RESULTS AND DISCUSSION The ability of different extracts from Rhubarb roots to scavenge DPPH (1,1-diphenyl-2-picrylhydrazyl) free Quantitative Analysis 22 radical was determined according to the method of Ye. Total Tannin Content of Rhubarb Roots Extract Briefly, a 0.1 mM of ethanolic DPPH solution was The results presented in Table 1 showed that, the prepared, to give the initial absorbance value of 0.993 at Rhubarb roots extract contain total tannin ranged from 517 nm. The different concentrations of Rhubarb extracts 559.81mg/100g DW (water extract) to 1248mg/100g DW (in 0.1 ml) were added to 3.0 ml of ethanolic DPPH International Journal of Pharmaceutical Sciences Review and Research International Journal of Pharmaceutical Sciences Review and Research Available online at www.globalresearchonline.net 94 Available online at www.globalresearchonline.net © Copyright protected.