Original Article In vitro Antioxidant and Free Radical Scavenging Activity of Extracts of Rosa damascena Flower Petals. Priti S. Patil1, Pratima A. Tatke*1 and Satish Y. Gabhe2

1Department of Pharmaceutical Chemistry C. U. Shah College of Pharmacy, S.N.D.T Women’s University, Juhu Campus, Santacruz (West), Mumbai 400 049, India 2Bharati Vidyapeeth Deemed University, Poona College of Pharmacy, Erandwane, Pune 411 038, India

ABSTRACT

Introduction: Rosa damascena mill L., well-known as Rose, has numerous pharmacological properties including antioxidant, antitussive, relaxant effect on tracheal chains, hypnotic, antibacterial and anti-HIV. Objective: The objective of the present study was to evaluate antioxidant and free radical scavenging activity of various extracts of Rose flower petals. Methods: Chloroform, acetone, ethanol, and aqueous extracts of fresh and dried Rose petals were prepared and evaluated for presence of flavonoids and phenolics. Ethanol and acetone extract showed high phenolic and flavonoid content and were selected for various in vitro free radical scavenging assays using DPPH scavenging, ABTS, peroxynitrite radicals, xanthine oxidase inhibition (XOI), superoxide scavenging and Lipid peroxidation assay. Results: All the extracts of fresh and dried Rose petals were found to Address for contain higher amount of total phenolics expressed as mg of GAE/g Correspondence of dry extract and total flavonoids expressed as mg of RE/g of dry extract. The amount of both these constituents was highest in ethanol Department of extract followed by acetone extract, aqueous extract and least in Pharmaceutical chloroform extract. The ethanol extract of fresh and dried petals Chemistry C. U. Shah showed lowest IC50 values than the acetone extract when evaluated College of Pharmacy, by DPPH, ABTS, peroxynitrite inhibition, xanthine oxidase S.N.D.T Women’s inhibition, superoxide scavenging and lipid peroxidation inhibition University, Santacruz methods indicating good free radical scavenging activity. (West), Mumbai 400 Conclusion: The present study has demonstrated that ethanol extract 049, India of fresh Rose flower petals exhibit potent antioxidant and free radical scavenging activities. E-mail: [email protected] Keywords: Rosa damascena, total phenolics, flavonoids, rutin, DPPH, ABTS.

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INTRODUCTION Free radicals are known to be the therapy of rose oil is helpful for several major cause of various chronic and allergies, migraine and headaches. degenerative diseases, including diabetes Numerous components were isolated from mellitus, inflammation, stroke, cancer, petals, flowers and hips (seed-pot) of R. coronary heart disease, and aging1,2. damascena including flavonoids, Reactive oxygen species (ROS) include free anthocyanins, glycosides, and terpenes. radicals such as .O2- (superoxide anion), Plant shows presence of kaempferol, .OH (hydroxyl radical), H2O2 (hydrogen carboxylic acid, quercetin, myrcene, and peroxide) and .O2 (singlet oxygen) which vitamin C. Flowers show presence of fatty cause cellular injuries and initiate oil, bitter principle, organic acids and peroxidation of polyunsaturated fatty acids tanning matter7.The essential oil of Rose in biological membranes3,4. The tissue injury contains more than 95% of total oil caused by ROS include protein damage, represented by eighteen compounds. β- DNA damage and oxidation of important citronellol (14.5-47.5%), geraniol (5.5- enzymes in the human cells. The 18%), nerol, kaempferol and nonadecane consequences of these events may lead to (10.5-40.5%) are the identified oils. In the occurrence of various oxidative stress addition, citrenellol (9.91%), phenyl ethyl related diseases. Recently, plants, natural alcohol (78.38%), geraniol and nonadecane foods and food derived antioxidants such as (4.35%) are also found to be present in Rose vitamins and phenolic phytochemicals have oil8. received growing attention, since they play an important role as preventive agents MATERIALS AND METHODS against damage caused due to oxidative stress5. Procurement of plant material Rosa genus, belonging to the Fresh petals of Rose flower were Rosaceae family, includes 200 species and procured from local market at Dadar, more than 18,000 cultivars. One of the most Mumbai, India and were authenticated at important Rosa species is Rosa damascena Agarkar Research Insitute, Pune, India. Mill. This plant is called damask rose because it was originally brought to Europe Chemicals and instruments from Damascus6. The essential oil of Rosa 1,1-Diphenyl-2-picrylhydrazyl damascena is known for its fine perfumery (DPPH), Gallic acid, 6-Hydroxy-2, 5, 7, 8- applications and the use in cosmetic tetramethylchroman-2-carboxylic acid preparations. The use of Rosa damascena is (Trolox), 2,2’-azinobis-(3-ethylbenzo- increasing steadily worldwide for its thiazoline)-6-sulphonic acid (ABTS), medicinal properties health promoting Allopurinol, Bovine Brain Extract (BBE), benefits. Recently, the antioxidant, Bovine Serum Albumin (BSA), were antibacterial, anti-HIV and activities of its procured from Sigma Chemical Co. (USA). essential oil have been demonstrated. This Xanthine oxidase, Nitroblue tetrazolium plant is also used as a gentle laxative. Rose chloride (NBT) and xanthine were procured oil is used for healing nervous stress, from Himedia Ltd. Mumbai, India. Folin- tension, grief and depression. It helps in the Ciocalteu solution, Pyrogallol Red, reduction of thirst, curing old cough, treating potassium dihydrogen phosphate and special complaints of women, wound thiobarbituric acid, dipotassium hydrogen healing, and improving skin health. Vapour phosphate, trichloroacetic acid, anhydrous

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sodium carbonate, ascorbic acid, butylated made upto 10 ml. The final mixture was hydroxytoluene (BHT) potassium persulfate, allowed to stand at room temperature for 2 h ferrous ascorbate, ethylene diamine tetra with intermittent shaking. Then the acetic acid (EDTA), were purchased from absorbance of the dark blue colour that S.D.Fine Chemicals, Mumbai, India. All developed was measured at 725 nm using other chemicals and solvents used were of UV-Vis spectrophotometer. Gallic acid was analytical grade. used as standard for preparing the The instruments used for the study calibration curve (10 μg/ml - 100 μg/ml). were UV spectrophotometer, (Jasco, V-630), The concentration of total phenolic laboratory centrifuge (Remi motors, R4C) compounds in the extracts was calculated and digital pH meter (Equip-tronics, EQ- using the following linear equation based on 610). the calibration curve: y = 0.010x + 0.01 with R2= 0.994. The total phenolic content in the Preparation of extracts plant extract was expressed as mg of gallic Fresh petals of Rose were acid equivalent per g of dry weight (mg immediately used for extraction. Some of GAE/g) of extract. All the tests were carried the fresh petals were dried, stored in air-tight out in triplicate and the results are expressed container and then used for extraction. The as mean ± SD. extraction of plant material was carried out by Soxhlet extraction method using various Quantification of total flavonoids solvents such as chloroform, acetone, The total flavonoid content of all the methanol. and water. All extracts were extracts was determined by aluminium filtered individually and further evaporated chloride colorimetric method9. 0.5 ml of to get dry extracts. The extractive values sample solution was mixed with 2 ml of were determined. After drying, crude distilled water and subsequently with 0.15 extracts were stored in desiccator till further ml of 5% NaNO2 solution. After 6 min of use. incubation, 0.15 ml of 10% AlCl3 solution was added and then allowed to stand for 6 Phytochemicals screening min, followed by adding 2 ml of 4% NaOH Extracts were qualitatively evaluated solution to the mixture. Immediately water for the presence or absence of active was added to the sample to make the final principles such as phytosterols, tannins, volume to 5 ml, the mixture was thoroughly flavonoids, saponins, alkaloids, glycoside, mixed and allowed to stand for another 15 triterpenoids and proteins. min. The absorbance was determined at wavelength 510 nm. The total flavonoid Quantification of total phenolics – by Folin- content was expressed in mg of rutin Ciocalteu method equivalent per g of extract (mg RE/g) of The total phenolic content in various extract. extracts of the plants were measured using Folin-Ciocalteu reagent9. 1 ml of extract In vitro antioxidant studies solution was added to 0.5 ml of Folin The ethanol and acetone extracts of Ciocalteau reagent and 5 ml of distilled fresh and dried Rose flower petals with high water. The mixture was incubated at room phenolic and flavonoid content were further temperature for 10 min. Then 1.5 ml of evaluated for their antioxidant and radical anhydrous sodium carbonate solution (10% scavenging potential w/v) was added and the final volume was

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DPPH (1, 1, diphenyl 2-picryl hydrazyl) The % inhibition of pyrogallol red bleaching assay was determined using the formula [(A1- The free radical scavenging activity A2)/A1] X 100, where A1 is the absorbance of the extracts, based on the scavenging in presence of antioxidants and A2 is the activity of the stable 1, 1-diphenyl-2- absorbance in absence of antioxidants. The picrylhydrazyl (DPPH) free radical was IC50 values of extracts yielding 50% determined10,11. A 0.1 mM solution of DPPH inhibition of pyrogallol red bleaching were in methanol was prepared. 1 ml of the estimated. Ascorbic acid was used as extract solution in various concentration standard antioxidant for comparison. All the ranges was added to 3 ml of the DPPH tests were carried out in triplicate and the solution. The decrease in absorbance was results were expressed as mean ± SD12-14. determined at 517 nm after 30 min. The percentage scavenging activity was ABTS assay calculated from [(A0–A1)/A0] × 100, where The antioxidant activities of all the A0 is the absorbance of the control, and A1 is extracts in the reaction with the stable the absorbance of the extract/ standard. A ABTS+• radical cation were blank is the absorbance of the control determined15,16,17. The reaction between reaction (containing all reagents except the ABTS and potassium persulfate generates test compound). The % scavenging activity the blue/ green ABTS+. chromophore, which and IC50 value of extracts were calculated can be reduced by an antioxidant, thereby for the various concentrations. Ascorbic acid resulting in decrease of absorbance at 734 was used as standard antioxidant for nm. ABTS was dissolved in water to a 7 comparison. All the tests were carried out in mM concentration. ABTS radical cation triplicate and the results were expressed as (ABTS+.) was produced by reacting ABTS mean ± SD. stock solution with 2.45 mM potassium persulfate and allowing the mixture to stand Inhibition of pyrogallol red bleach by in the dark at room temperature for 12–16 peroxynitrite hr. The ABTS+. solution was diluted with a Peroxynitrite (ONOO-) was prepared phosphate buffer (2 mM, PH 7.4) to achieve by reacting 2 M H2O2 in 2 M HNO3 with 2 an absorbance of 0.8 ±0.014 at 734 nm. +. M NaNO2, followed by stabilization of the Extract solutions were mixed with ABTS product with 4 M NaOH. The solution was solution, and the absorbance was read after 1 frozen at -70ºC. Peroxynitrite concentration min using UV-vis spectrophotometer at 734 was determined spectrophotometrically at nm. Phosphate buffer solution was used as a 302 nm (ε=1670 M-1 cm-1) and dilutions in blank. The % radical-scavenging activity of 1M NaOH were made in order to achieve the samples was determined using the 200 µM solution. All the extract solutions formula [(Acontrol–Atest)/ Acontrol] X 100, were prepared by dissolving in methanol. where Acontrol is the absorbance of the 100 µM Pyrogallol red solution was control (ABTS+• solution without test prepared in 100 mM phosphate buffer, pH sample) and Atest is the absorbance of the 7.4. 1 ml of extract solution was added to 2 test sample (ABTS+• solution with extract). +. ml of 100 µM pyrogallol red solution.0.5 ml The IC50 values scavenging 50% of ABTS of 200 µM peroxynitrite solution was added were estimated. Ascorbic acid and trolox to the mixture and immediately vortexed. were used as standard antioxidants for After 15 min the absorbance was measured comparison. All the tests were carried out in using UV-vis spectrophotometer at 540 nm.

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triplicate and the results were expressed as the amount of the formazan which was mean ± SD. reduced from NBT by superoxide using the following equation where, A1- Absorbance Xanthine oxidase assay of control and A2- Absorbance in presence The inhibitory effect on xanthine of test compounds: % Inhibition= [A1- oxidase was measured spectro- A2/A1 X 100] photometrically at 295 nm18,19. The reaction mixture consisted of 50 mM sodium Ascorbate iron induced lipid peroxidation phosphate buffer (pH 7.5), sample solution 10 mg of bovine brain extract was dissolved in distilled water or DMSO, mixed with phosphate buffer pH 7.4 and freshly prepared enzyme solution (0.2 U/ml sonicated in an ice bath until a milk-like of xanthine oxidase in phosphate buffer) and suspension was obtained. The lipid distilled water. The assay mixture was pre- suspension was mixed with 1 mM FeCl3 and incubated at 37°C for 15 min. Then extract. The peroxidation was initiated by substrate solution (0.15 mM of xanthine) adding 1 mM ascorbate. The mixture was was added into the mixture. The mixture incubated at 370C for 60 min. After was incubated at 370C for 30 min. Next, the incubation, 10% trichloroacetic acid was reaction was stopped with the addition of 0.5 added and centrifuged at 1800 rpm for 10 M HCl. The absorbance was measured using minutes. After centrifugation, 1ml of UV-VIS spectrophotometer against a blank supernatant was collected and mixed with prepared in the same way but the enzyme 1ml of 0.67% thiobarbituric acid (TBA). solution was replaced with the phosphate The mixture was vortexed and heated in buffer. Another reaction mixture was boiling water bath at 1000C for 20 min and prepared (control) having water/DMSO then rapidly cooled and the extent of instead of test compounds in order to have inhibition of peroxidation was estimated maximum uric acid formation. Thus, XOI from the absorbance of the upper organic activity was calculated using the following layer at 532nm. A tube containing all the equation in which α is the activity of XO reaction mixture except the plant extract was without test extract and β is the activity of used as control. Blank was phosphate buffer. XO with test extract. % XO inhibition = (1 – The percent inhibition was calculated with β/α) x 100 the formula Percent inhibition (%) = (Abs of control – Abs of sample) X 100 / (Abs of Superoxide scavenging assay control)23. Superoxide was generated by xanthine oxidase system20-22. The reaction RESULTS mixture consisted of sodium phosphate buffer (pH 7.4), 3 mM Xanthine, 3 mM Extractive values EDTA, 0.15%BSA, 15 mM Nitroblue The extractive values of fresh rose tetrazolium chloride, sample solution The petals were found to be higher as compared reaction mixture was incubated at 250C for to dried rose petals. The extractive values 10 min. Then reaction was initiated by for fresh and dried petals of Rose flower adding 1.5 U/ml of xanthine oxidase and were highest for aqueous extract (32.68, incubated at 250C for 20 min.After 20 min 27.90% w/w respectively) followed by the absorbance was measured at 560 nm ethanol extract (19.21, 15.62 % w/w using UV-Vis spectrophotometer.The respectively), acetone extract (12.94, 8.65% inhibtion rate was calculated by measuring w/w respectively) and chloroform extract (4.56, 1.10 % w/w respectively).

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Quantitation of total phenolics and total Rose petal extracts exhibited the activity at flavonoids higher IC50 values from 92.02±0.86, Ethanol extract and acetone extract 94.09±2.41, 96.14±0.73and 104.81±3.40 of the fresh petals of Rose were found to µg/ml respectively as compared to standard contain higher amount of total phenolics ascorbic acid (IC50 = 49.41±0.27µg/ml) (147.1, 129.73 mg GAE/g of extract) and total flavonoids (161.36, 141.5 mg RE/g of ABTS radical scavenging assay extract) as compared to other extracts Ethanol extracts and acetone extracts (Figure.1). Ethanol and acetone extract of of fresh and dried Rose petals exhibited dried Rose petals were found to contain high ABTS scavenging activity at higher IC50 amount of total phenolics (77.37, 66.58 mg values ranging from 325.36±4.53, GAE/g of extract) and total flavonoids 416.75±2.78, 554.77±4.79 and 631.00±9.36 (116.92, 114.97 mg RE/g of extract as µg/ml respectively as compared to standard compared to other extracts. Therefore, these ascorbic acid 19.03±0.08 µg/ml and trolox extracts were selected for various in-vitro 9.34±0.08 µg/ml (Table.1 and Table.2). antioxidant assay methods. Xanthine oxidase inhibition assay In vitro antioxidant assays Allopurinol is a potent inhibitor of xanthine oxidase. It inhibited the enzyme at DPPH scavenging IC50 value of 3.38 ± 0.16. Ethanol and Both ethanol and acetone extracts of acetone extracts of fresh Rose petals fresh and dried rose petals were effective in inhibited the enzyme at higher IC50 values of reducing the stable radical DPPH to the 91.59±1.22 to 115.85±1.46 µg/ml yellow colored diphenylpicrylhydrazine, respectively. Also, ethanol and acetone indicating that these extracts were able to extracts of dried Rose petals inhibited the scavenge DPPH radical (Table.1 and enzyme at still higher IC50 values of 129.32 Table.2). Standard ascorbic acid exhibited ±0.80 to 157.92±0.80 µg/ml respectively maximum free radical scavenging activity (Table.1 and Table.2). with IC50 =13.24 ±0.48µg/ml. Ethanol extract of Rose fresh petals (IC50 = Superoxide scavenging assay 19.72±0.20 µg/ml) demonstrated a stronger Superoxide radical scavenging DPPH radical scavenging activity than activity was shown by both ethanol and acetone extract (IC50 = 25.75±1.49 µg /ml). acetone extract of fresh and dried Rose Dried petals ethanol extract (IC50 = petals. Allopurinol is a potent scavenger of 21.97±0.05 µg/ml) showed good DPPH superoxide anions with IC50 value of scavenging as compared to acetone extract 6.00±0.23 µg/ml. The superoxide (IC50 = 29.97±1.66 µg/ml). scavenging effect for fresh rose petals was found to be greater for ethanol extract (IC50 Peroxynitrite inhibition 696.59±1.44 µg/ml) as compared to acetone Pyrogallol Red is a dye which gets extract (IC50 716.46±3.82 µg/ml). Also the bleached by peroxynitrite radical. All the scavenging effect for dried rose petals was rose petal extracts moderately inhibited found to be greater for ethanol extract (IC50 bleaching of peroxynitrite as compared to 823.85±7.64 µg/ml) followed by acetone standard ascorbic acid (Table.1& Table.2). extract (IC50 888.32±2.56 µg/ml) (Table.1 Ethanol and acetone extracts of fresh Rose and Table.2). petal, ethanol and acetone extract of dried

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Lipid peroxidation This study reports the various in Rose fresh petals ethanol and vitro antioxidant and free radical scavenging acetone extracts inhibited lipid peroxidation activities of the extracts of Rose petals at higher IC50 values ranging from which may be associated with its health 110.42±8.05to 142.32±2.75 µg/ml as benefits. Rose petal extracts showed compared to butylated hydroxyl toluene antioxidant activities, however, the 33.11±1.44 µg/ml. Also, Rose dried petals magnitude of antioxidant potency varied ethanol and acetone extracts inhibited lipid with the type of extracts. This could be due peroxidation at still higher IC50 values to the difference in concentrations and type ranging from 134.42±4.00 to of antioxidant compounds present in these 167.94±4.01µg/ml (Table.1 and Table.2). extracts. The broad range of antioxidant activity of the extracts indicates the potential DISCUSSION of the Rose flower petals as a source of natural antioxidants. It can have potential Plant cells contain a wide variety of application to reduce oxidative stress with phytoconstituents with antioxidant activity. consequent health benefits. The healing properties of medicinal plants DPPH solution is decolourised from are mainly due to the presence of various deep violet to light yellow. The degree of secondary metabolites such as phenols, reduction in absorbance measurement is flavonoids, glycosides, saponins, sterols, indicative of the radical scavenging alkaloids, etc. Ployphenolic compounds such (antioxidant) power of the extract. IC50 as flavonoids and tannins, nitrogen (concentration required to obtain a 50% compounds such as alkaloids, amino acids antioxidant capacity or is the concentration and amines), lignans, carotenoids, terpenes of substrate that brings about 50% loss of possess antioxidative activity by suppressing the DPPH) is typically employed to express the initiation or propagation of the chain the antioxidant activity and to compare the reactions. The preliminary phytochemical antioxidant capacity of various samples25. tests revealed the presence of carbohydrates, Both the ethanol and acetone extract of fresh flavonoids, phenols, carbohydrates, and dried Rose petals exhibited good saponins, sterols and tannins in both the activity comparable to standard ascorbic Rose fresh petals and dried petals extracts. acid. The preliminary screening tests are thus Pyrogallol red is a dye. Peroxynitrite useful in the detection of the bioactive radical bleaches this dye and the intensity of principles and subsequently may lead to the dark red colour decreases. Antioxidants drug discovery and development. scavenge the peroxynitrite radical thereby In order to evaluate antioxidant preventing the bleaching of pyrogallol red potential of a plant, it is desirable to subject and retaining its colour intensity. In this it to a series of tests that evaluate the range assay, the increase in absorbance is of activities such as scavenging of the proportional to inhibition of pyrogallol red reactive oxygen species, inhibition of bleach by peroxynitrite26. Extracts of fresh membrane lipid peroxidase and enzyme as well as dried Rose petals were found to inhibition. Plant extracts rich in antioxidants have appreciable peroxynitrite scavenging serve as source of nutraceuticals that activity. alleviate the oxidative stress and therefore ABTS.+ radical is a chromophore prevent or slow down the degenerative 24 having blue colour. The selected extracts diseases . were able to scavenge the radical cation

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effectively at higher IC50 values as petals extracts exhibited a better antioxidant compared to standards. action against peroxidation of Bovine brain Xanthine oxidase is a flavoprotein, extract, suggesting better cell membrane which catalyses the oxidation of protection against lipid proxidation. An hypoxanthine to xanthine and generates increase in oxidative stress is widely superoxide and uric acid Studies have shown accepted as one of the main factors that xanthine oxidase inhibitors may be involving in the development of peroxyl useful for the treatment of hepatic disease radicals, all of which may play a role in and gout, which is caused by the generation DNA damage, glycation, and protein of uric acid and superoxide anion radical. modification reactions, and in lipid oxidative Xanthine oxidase-derived superoxide anion modification in diabetes. has been linked to various degenerative and metabollic disorders. Superoxide radicals CONCLUSIONS are generated during the normal physiological process mainly in In conclusion, the present study has mitochondria. Although superoxide anion is demonstrated that ethanol extract of fresh by itself a weak oxidant, it gives rise to the Rose flower petals exhibit potent antioxidant powerful and dangerous hydroxyl radicals as and free radical scavenging activities. The well as singlet oxygen both of which activity could be derived from compounds contribute to the oxidative stress. Therefore such as flavonoids and phenolics which are superoxide radical scavenging by polar phytoconstituents. These phyto- antioxidants has physiological implications. constituents are well known antioxidants Allopurinol is a potent inhibitor of xanthine and free radical scavengers. These oxidase. However chronic treatment with antioxidant activities can be at least partly allopurinol has its own side effects. Hence, linked to the therapeutic benefits of the natural antioxidants which inhibit this certain traditional claims of Rose flower enzyme and also have superoxide petals. scavenging activity would be beneficial in preventing various complications of ACKNOWLEDGEMENT oxidative stress related disorders. Rose The authors acknowledge University flower petals were found to have moderate Grants Commission (UGC) for financial xanthine oxidase inhibitory activity as well support to carry out the research work. as superoxide scavenging activity though at

higher IC values as compared to 50 Conflict of interest allopurinol. But the safety of this drug along The authors hereby declare no with its antioxidant potential will be a better conflict of interest for the research work. therapeutic approach27,28.

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Table-1: IC50 values of Rose fresh petal extracts for various free radical scavenging activity

IC50 values of extracts (µg/ml) Rose fresh petals Extract Xanthine Superoxide Lipid DPPH Peroxynitrite ABTS oxdiase Scavenging Peroxidation Ethanol 19.72±0.20 92.02± 0.86 325.56±4.53 91.59±1.22 696.59±1.44 110.42±8.05 Acetone 25.75±1.49 94.09±2.41 416.75±2.78 115.85±1.46 716.46±3.82 142.32±2.75 Ascorbic Acid 13.24±0.48 49.41±0.27 19.03±0.08 - - - Trolox - - 9.34±0.08 - - - Allopurinol - - - 3.38 ± 0.16 6.00±0.23 - BHT - - - - - 33.11±1.44

Table- 2: IC50 values of Rose dried petal extracts for various free radical scavenging activity

IC50 values of extracts (µg/ml) Rose dried petals Extract Xanthine Superoxide Lipid DPPH Peroxynitrite ABTS oxdiase Scavenging Peroxidation Ethanol 21.97±0.05 96.14±0.73 554.77±4.79 129.32±0.80 823.85±7.64 134.42±4.00 Acetone 29.97±1.66 104.81±3.40 631.00±9.36 157.92±1.30 888.32±2.56 167.94±4.01 Ascorbic Acid 13.24±0.48 49.41±0.27 19.03±0.08 - - - Trolox - - 9.34±0.08 - - - Allopurinol - - - 3.38 ± 0.16 6.00±0.23 BHT - - - - - 33.11±1.44

AJPCT[3][09][2015] 589-601 Tatke et al______ISSN 2321 – 2748

Fig. 1: Calibration Curve for Standard Gallic Acid

Fig. 2: Gallic Acid Equivalent of Rose fresh petals and dried petals

Values are presented as mean ± SD (n = 3).

AJPCT[3][09][2015] 589-601 Tatke et al______ISSN 2321 – 2748

Fig. 3: Calibration Curve for Standard Rutin

Fig. 4: Rutin Equivalent of Rose fresh petals and dried petals

Values are presented as mean ± SD (n = 3).

AJPCT[3][09][2015] 589-601