International Journal of Life-Sciences Scientific Research (IJLSSR), VOLUME 1, ISSUE 2, pp: 74-78 NOVEMBER-2015 Research Article (Open access) Antioxidant and Antibacterial Activity of Jurinea dolomiaea Boiss Extracts Pratap Singha*, Rajendra Singha, Nitin Satib, Om Prakash Satia, Naresh Kumara a b Department of Chemistry, HNB Garhwal University (A Central University), Srinagar Garhwal, Uttarakhand, India. Department of Pharmaceutical Sciences, HNB Garhwal University (A Central University), Srinagar Garhwal, Uttarakhand, India ABSTRACT- The aim of this study was to investigate in vitro antioxidant activity and anti-bacterial activity of the petroleum ether, ethyl acetate and methanol extract obtained from the whole part of Jurinea dolomiaea Boiss (Asteraceae). Total phenolic and flavonoid contents of these extracts were determined as gallic acid and rutin equivalents, respectively. Total antioxidant activity, reducing power of these extract were evaluated as ascorbic acid and gallic acid equivalents, respectively. ABTS free radical scavenging activity is expressed as trolox equivalent antioxidant capacity (TEAC). The antibacterial activity of the extract was investigated by disc diffusion method. The ethyl acetate and methanol extracts showed moderate activity against E. coli and S. aureus. Key words: Jurinea dolomiaea; Total phenolic; Total flavonoid; Total antioxidant; Free radical scavenging activity; Antibacterial activity. -------------------------------------------------IJLSSR----------------------------------------------- 1. INTRODUCTION Jurinea dolomiaea Boiss syn Jurinea macrocephala Royle is rosette- The germacranolides oxygenated at C-14 and C-15are characteristic for forming stemless perennial herbs. Leaves flat on the ground, to about 15- this genus (Rustaiyan A and Ganji M 1988). 45cm long, pinnate to pinnatisect, the leaflets again lobed or toothed, The pentacyclic triterpenes were also found in root of Jurinea albicaulis white woolly beneath, mid to grey-green above with a reddish midrib, (Todorova M and Ognyanov I 1996). Jurinea species are reported to be achenes, ashy grey. Flowerheads purple, 2-3cm across, up to thirty in a useful as antimicrobial Activity (Kirbag S et al., 2009), antioxidant activ- central domed cluster in the centre of each rosette, late summer to early ity and anticholinesterase Activity (Ozturk H et al., 2011). Jurinea dolo- autumn, Pakistan to eastern Nepal, on open slopes at 4000m (Naithani miaea have been previously reported antibacterial activity (Dwivedi SD BD 1984). Plant is used in Indian traditional system of medicine. A de- and Wagay SA 2014), and antioxidant activity (Shah NA et al., 2014). coction of the root is cordial; it is given in the treatment of colic and 2. Materials and Methods puerperal fever & the bruised root is applied as a poultice to eruptions 2.1. Plant collection and identification: (Chopra RN et al., 1986). The plant is used in Nepal for incense and the Fresh plant material of Jurinea dolomiaea Boiss were collected in Octo- juice of the roots is used in the treatment of fevers (Manandhar NP ber 2012 from the North-West Himalayas, Uttarakhand, India and identi- 2002). fied from Forest Research Institute, Dehradun, India to authentic sample. The limited phytochemical work on Jurinea species revealed that their 2.2. Extract preparation: main constituent was the sesquiterpene lactones (Rustaiyan A et al., The whole plant of J. dolomiaea was dried at room temperature (25̊ C). Correspondence details The dried sample was chopped into small parts with a blender. Powder *Pratap Singh (2 kg) was extracted successively with petroleum ether, ethyl acetate and Research Scholar methanol in soxhlet apparatus for 24 h. The extracts were filtered over Department of chemistry filter paper and the organic solvent extracts were concentrated under HNB Garhwal University (A Central University) vacuum using rotary evaporator and the crude extract was obtained, Srinagar Garhwal (246 174), Uttarakhand, India separately. Email: pratapsinghbishtb4@gmail.com 2.3. Determination of total phenolic content: Phone: +91-7536877288 1981; Todorova M and Ognyanov I 1984; Zakirov SK et al., 1975). Total phenols were determined by a Folin-Ciocalteu method (Wolfe G Received: 08 October 2015/Revised: 17 October 2015/Accepted: 29 October 2015 working Folin-Ciocalteu solution, 0.5 ml extracts and 2 ml of saturated et al., 2003). The measurement was conducted by mixing 2.5 ml of sodium carbonate solution. The absorbance was measured after 30 mi- http://ijlssr.com IJLSSR © 2015 All rights are reserved International Journal of Life-Sciences Scientific Research (IJLSSR), VOLUME 1, ISSUE 2 nutes at 765 nm, along with the blank. The standard gallic acid diagram tion with 2.4mM potassium persulfate in the dark for 16h at room tem- was prepared by adding gallic acid of different concentration instead of perature (Roberta et al. 1999). Prior to assay, the solution was diluted in 0.5 ml of sample. The total phenolic content was calculated as phenols ethanol and equilibrated at 30°C to give an absorbance of 0.700±0.02 at equivalent to gallic acid (mg GAE/g). 734nm. The stock solution of the sample extracts were diluted such that 2.4. Determination of total flavonoid content: after introduction of 10µL aliquots into the assay, they produced between Total flavonoids in the plant extracts were estimated by using the me- 20% and 80% inhibition of the blank absorbance. After the addition of thods (Patel et al., 2011). The extract (500µL) was diluted appropriately 1ml of diluted ABTS solution to 10µL of sample or Trolox standards in and mixed with 1ml NaNO2 (5%). After standing for 6min, 1ml of 10% ethanol, absorbance was measured exactly 30min after the initial mixing. AlCl3 and 10ml of NaOH (1M) were added to the mixture. The mixture Appropriate solvent blanks were also run in each assay and the percen- was adjusted to 25ml with 70% ethanol and allowed to rest for 15min. tage inhibition of the blank absorbance was calculated at 734nm. Tripli- The absorbance was measured at 510nm, with 70% ethanol as a blank cate determinations were made at each dilution of the standard and per- control. Total flavonoid content was estimated by using a calibration centage inhibition calculated and plotted as a function of Trolox concen- curve of rutin and expressed as mg rutin equivalents per g of sample tration. The antioxidant activity is expressed as trolox equivalent anti- (mgRE/g). oxidant capacity (TEAC). 2.5. Total antioxidant capacity: 2.9. Anti-bacterial activity: Sample (0.3ml) was mixed with 3.0ml reagent solution (0.6M sulfuric Standard and isolated strains of bacteria used to test antibacterial activi- acid, 28mM sodium phosphate and 4mM ammonium molybdate). Reac- ties of the extracts are given in (Table 2). Antibacterial activity was eva- tion mixture was incubated at 95°C for 90 min under water bath. Absor- luated by the disc diffusion method (Ahluwalia et al., 2014) with slight bance of all the sample mixtures was measured at 695nm (Prieto, Pineda modification against gram-positive and gram-negative bacteria. Bacteria & Aguilar 1999). Total antioxidant capacity was expressed as ascorbic were obtaind from the Microbial Type Culture Collection, Institute of acid equivalent per gram extract (mgAAE/g). Microbial Technology, Chandigarh (India). 2.6. Reducing power: Disc diffusion assay The reducing power of Extracts was determined according to the me- Nutrient agar medium (20 mL) was poured into the plates to a uniform thods (Oyaizu M 1986). Different concentration of extracts (50- depth and allowed to solidify. The standard inoculum suspension (106 500µg/ml) in 1 ml of extracts in 1ml of alcohol was mixed with 2.5ml c.f.u. /ml) was streaked over the surface of the media using a sterile phosphate buffer (0.2M, pH6.6) and 2.5ml of 1% potassium ferricyanide. cotton swab to ensure the confluent growth of the organism. Plant The mixture was incubated at 50°C for 20min and 2.5ml of 10% trichlo- extract (10 µL) was diluted with two volumes of 5% dimethyl sulfoxide, roacetic acid was added. The reaction mixture was then centrifuged for impregnated on filter paper discs, and used for the assays. On the surface 10min. Further, 2.5ml of the supernatant solution was mixed with 2.5ml of the plates, discs were placed with sterile forceps, pressed gently to of distilled water and 0.5ml of 0.1% FeCl3. The absorbance was meas- ensure contact with the inoculated agar surface. Oxacillin (10 µg disc-1) ured at 700 nm. was used as a positive control and hexane as a negative control. The 2.7. DPPH free radical scavenging activity: plates were incubated in the dark at 37 oC (24 h) and the inhibition zones The free radical scavenging activity of extracts was measured by 1,1- calculated. All experiments were carried out in triplicate. diphenyl-2-picryl-hydrazil (DPPH˙) using the method (Sheng et al. 3. RESULTS AND DISCUSSION 2007). A 2ml aliquot of solution was added to 2ml of 2x10-4mol/L etha- 3.1. Total phenolic contents: nolic DPPH solution. The mixture was shaken vigorously and the absor- Phenolic compounds such as flavonoid, tannins and phenolic diterpenes bance was measured at 517nm immediately. The decrease in absorbance possess antioxidant activity. Folin-Ciocalteu method of determination of was determined at 15 and 30min until the absorbance reached a steady total phenolic content is based on the principle that oxidation of phenol state (after nearly 30 min). The mixture with the addition of standard by molybdotungstophosphoric reagent yield a colored product that is antioxidants served as a positive control. All the tests were performed in estimated by measuring absorbance at 765nm. Gallic acid was used as triplicate, and the inhibition rate was calculated according to the formula, reference standard and the phenolic contents of the extracts were ex- % Inhibition of DPPH free radical = [(A blank – A sample) / A blank] x 100 pressed in mg Gallic acid equivalents per gram of extract (Table 1). The 2.8. ABTS free radical scavenging activity: (295.36 ± 2.88 mg GAE/g) followed by methanolic extract (193.3 ± 4.40 ABTS free radical was produced by reacting 7mM ABTS aqueous solu- mg GAE/g) and the petroleum ether extract (78 ± 1.1547 mg GAE/g) of highest amount of phenolic content was found in the ethyl acetate extract J. dolomiaea. http://ijlssr.com IJLSSR © 2015 All rights are reserved International Journal of Life-Sciences Scientific Research (IJLSSR), VOLUME 1, ISSUE 2 3.2. Total flavonoid contents: antioxidant demonstrated significant reducing power far better than the Flavonoids possess a wide range of bioactivities including antioxidant extracts and rutin. The results indicate that the methanolic extract of J. activity. The presence of hydroxyl groups in the chemical structure of dolomiaea has a fair ability to act as electron donor and convert free flavonoids is responsible for their antioxidant activity. The determination radicals to stable products. of total flavonoid content using aluminum chloride is based on the for- Note: PEJD, EAJD, MEJD and AA are petroleum ether, ethyl acetate, mation of stable complex between aluminum chloride and keto and hy- methanolic extracts and ascorbic acid of Jurinea dolomiaea respectively. droxyl groups of flavonoids. The total flavonoid content of the extracts 3.5. DPPH free radical scavenging ability: of J. dolomiaea is expressed as rutin equivalents in mg/g extract (Table The DPPH method is based on the ability of stable free radical 2,2- 1). The high amount of flavonoids in the extract indicated the possible diphenyl-picrylhydrasyl (DPPH) to react with hydrogen donors, includ- antioxidant potential of the extracts. The methanolic extract (200.18 ± ing phenol compounds. DPPH shows an intensive absorption in the visi- 5.773 mg RE/g) showed the presence of higher flavonoid contents. The ble part of the spectrum and is easily determined spectrophotometrically. high amount of flavonoids in the methanolic extract (200.18 mg/g), pe- The results of the assay demonstrated antioxidant activity of Jurinea troleum ether extracts (184.78 ± 3.42 mg RE/g) and ethyl acetate extract dolomiaea extracts suggesting that the extracts are capable of donating (162.96 ± 4.3588 mg RE/g) suggested the possible antioxidant potential hydrogen and acting as natural antioxidants. The potential to scavenge of the J. dolomiaea extracts. DPPH radical was measured by determining IC50 value which indicate 3.3. Total antioxidant capacity: the concentration required to inhibit 50% of DPPH free radicals. IC50 Total antioxidant capacity determination by phosphomolybdenum me- value of the ethyl acetate extract (93.07 µg/ml) was much higher than thod involves formation of a green phosphate/Mo5+ complex at acidic that of methanolic (102.2 µg/ml) and pet ether extracts (307 µg/ml) of J. pH and is measured by absorbance at 695nm. The total antioxidant ca- dolomiaea (Table 1). IC50 value of ethyl acetate extract was much higher pacity of the extracts of J. dolomiaea is expressed as ascorbic acid then methanolic extract and petolium ether extract. It means ethyl acetate equivalent (AAEmg/g extract). The calibration curve of standard ascor- extract is powerful antioxidant capability (Fig 2). bic acid standard solutions was used to determine the total antioxidant capacity of the extracts. The antioxidant capacity of petroleum ether, ethyl acetate and methanolic extracts are 51.3 mg/g, 156.9 mg/g and 119.34 mg/g respectively (Table 1). Ethyl acetate extract showed highest total antioxidant activity. 3.4. Reducing power: Reducing power of the extract is determined on the ability to reduce a yellow color Fe3+/ferric cyanide complex to form Fe2+ ferrous complex. The amount of Fe2+ was monitored by measuring the formation of FIG. 2: DPPH FREE RADICAL SCAVENGING ACTIVITY (IC50) OF JUR- blue color at 700nm. A higher value of absorbance implies higher con- INEA DOLOMIAEA EXTRACTS Note: PEJD, EAJD, MEJD, BHA and AA are petroleum ether, ethyl acetate, methanolic extracts, butylated hydroxy anisole and ascorbic acid of Jurinea dolomiaea respectively. 3.6. ABTS radical action scavenging assay: An antioxidant is added to preformed ABTS radical cation and after a fixed time period the remaining ABTS is quantified. The activity of the tested sample extracts is expressed as Trolox equivalent antioxidant capacity (TEAC) defined as micromolar Trolox solution having an antioxidant capacity equivalent to 1g extract. Trolox (6-hydroxy-2,5,7,8centration of Fe+2 complexes and indicates higher reducing power. The tetramethylchroman-2-carboxylic acid) a water soluble analog of vitamin methanolic extract demonstrated highest reducing power followed by E is used as standard to represent the antioxidant strength of sample. The extracts exhibited good ABTS radical scavenging ability as all of Fig. 1: Reducing power of Jurinea dolomiaea extracts them were capable of decolorizing the ABTS radical color. The ethyl ethyl acetate extract of J. dolomiaea (Fig 1). Ascorbic acid, a synthetic acetate extracts demonstrated highest Trolox equivalent activity http://ijlssr.com IJLSSR © 2015 All rights are reserved International Journal of Life-Sciences Scientific Research (IJLSSR), VOLUME 1, ISSUE 2 (Table 1) of 11036.62 whereas the methanolic extract (7087.89) and Table 2: Antibacterial activity of Jurinea dolomiaea extracts petroleum ether extract exhibited (3129.34). Table 1: Antioxidant potential of Jurinea dolomiaea extracts TPCA TFCB TAOCC DPPH ABTSD (MG GAE/G) (MG RE/G ) (MG AAE/G) IC50(µG/ML) (TEAC) PEJD 78 (115.47) 184.78 (3.42) 51.3 307 3129.34 EAJD 295.36(2.88) 162.96 (4.358) 156.9 93.07 11036.62 MEJD 193.3 (4.40) 200.18 (5.773) 119.34 102.2 7087.89 RUTIN - - - 45 - BHA - - - 10 - ASCORBIC ACID - - - 21 - EXTRACT/STANDARD Note: PEJD, EAJD and MEJD are petroleum ether, ethyl acetate and methanolic extracts of Jurinea dolomiaea respectively. Note: PEJD, EAJD and MEJD are petroleum ether, ethyl acetate and 4. CONCLUSION methanolic extracts of Jurinea dolomiaea respectively. BHA is butylated In conclusion, we found that the ethyl acetate fraction from Jurinea do- a hydroxy anisole; total phenolic contents (TPC) are expressed as gallic lomiaea had higher total phenolic content, total antioxidant activity, acid equivalent; btotal flavonoid contents (TFC) are expressed as rutin DPPH scavenging activity, and ABTS radical action scavenging activity. equivalent; ctotal antioxidant activity (TAOC) is expressed as ascorbic The higher total flavonoids contents and anti-bacterial activity observed d acid equivalent; TEAC is trolox equivalent antioxidant capacity defined in methanolic fraction. The petroleum ether extract of Jurinea dolomiaea as micromolar trolox solution having antioxidant activity equal to 1g did not show any anti-bacterial activity against E. coli MTCC-443, Sal- extract; values in parenthesis indicate SD (n=3) monella typhirium MTCC-1255, Klebsiella pneumoniae MTCC-432 and 3.7. Anti-bacterial activity: Staphylococcus aureus MTCC-737. However ethyl acetate and metha- Antibacterial Screening of different extracts of Jurinea dolomiaea was nolic extract showed moderate activity against E. coli and S. aureus. The carried out by disc diffusion method. The zone of inhibition (mm) of the results clearly indicate antioxidant ability of the polar extract of Jurinea extracts against four pathogenic bacterial strains E. coli MTCC-443, dolomiaea and potential anti-bacterial activity of its ethyl acetate and Salmonella typhirium MTCC-1255, Klebsiella pneumoniae MTCC-432 methanolic extract. The results of study also indicate the need for further and Staphylococcus aureus MTCC-737. The extract was found to be phytochemical investigation of ethyl acetate and methanolic extract of possessing antibacterial activity against some pathogenic bacteria (Table Jurinea dolomiaea. 2). Maximum zone of inhibition was found to be methanol extract (15 5. REFERENCES mm) against E. coli. The petroleum ether extract, ethyl acetate extract [1] Ahluwalia V, Kumar J, Sisodia R, Shakil NA, Walia S. (2014). Green syn- and methanol extract do not show activity against Salmonella typhirium thesis of silver nanoparticles by Trichoderma harzianum and their bio- and Klebsiella pneumoniae. Ethyl acetate extracts show antibacterial efficacy evaluation against Staphylococcus aureus and Klebsiella pneumonia. Industrial Crop and Products, 55, 202-206. activity against E coli (13 mm) and Staphylococcus aureus (10 mm). So from the above study it can be concluded that the methanol and ethyl [2] Plants (Including the Supplement). Council of Scientific and Industrial Re- acetate extract of Jurinea dolomiaea possess antibacterial activity against some bacterial strains. Chopra RN, Nayar SL, and Chopra IC. (1986). Glossary of Indian Medicinal search, New Delhi. [3] Dwivedi SD, Wagay SA. (2014). Antimicrobial activity of leaf extracts of Jurinea dolomiaea plant against clinical and phytopathogenic bacteria. Chemical and Process Engineering Research, 24, 9-13. [4] Kirbag S, Zengin F, Kursat M. (2009). Antimicrobial activity of extracts of [5] Manandhar NP. (2002). Plants and People of Nepal Timber Press. Oregon. [6] Naithani BD. (1984). Flora of Garhwal: Botanical Survey of India, Vol-1, some plants. Pak. J. Botany, 41, 2067-2070. 311. [7] Oyaizu M. (1986). Studies on product browning reaction prepared from glucose amine, Japan Journal of Nutrition, 44, 307-315. http://ijlssr.com IJLSSR © 2015 All rights are reserved International Journal of Life-Sciences Scientific Research (IJLSSR), VOLUME 1, ISSUE 2 [8] Ozturk H, Kolak U, Meric C. (2011). Antioxidant, anticholinesterase and antibacterial activities of Jurinea consanguinea DC. Rec. Nat. Product, 5(1), 43-51. [9] Patel DK, Kumar R, Prasad SK, Hemalatha S. (2011). Pedalium murex Linn. (Pedaliaceae) fruits: A comparative antioxidant activity of its different fractions. Asian Pacific Journal of Tropical Biomedicine. 395-400. [10] Prieto P, Pineda M, Aguilar M. (1999). Spectrophotometric quantification of antioxidant capacity through the formation of phosphomolybdenum complex: specific application to determination of Vitamin E. Annals of Biochemistry, 269, 337-341. [11] Roberta R, Nicoletta P, Anna P, Ananth P, Min Y, Catherine RE. (1999). Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radicals Biology and Medicine, 26, 1231-1237. [12] Rustaiyan A, Ganji M. (1988). Germacranolides from Jurinea eriobasis. Phytochemistry, 27, 2991-2992. [13] Rustaiyan A, Niknejad A, Bohlmann F, Schuster A. (1981). Naturally occuuring terpene derivatives. A guaianolide from jurinea carduiformis. Phytochemistry, 20, 1154. [14] Shah NA, Khan MR, Naz K, Khan MA. (2014). Antioxidant Potential, DNA Protection, and HPLC-DAD Analysis of Neglected Medicinal Jurinea dolomiaea Roots. Biomed Research International, 2014. [15] Sheng JC, Zhou J, Wang L, Xu J, Hu Q. (2007). Antioxidant activity of ethanol and petroleum ether extracts from Brazilian propolis. European Food Research and Technology, 225, 249-53. [16] Todorova M, Ognyanov I. (1984). Sesquiterpene lactones in leaves of Jurinea albicaulis. Planta Medica, 50, 452-453. [17] Todorova M, Ognyanov I. (1996). Pentacyclic triterpenes in root of Jurinea albicaulis. Fitoterapia, 67, 383. [18] Wolfe G, Wu X, Liu RH. (2003). Antioxidant Activity of Apple Peels. J. Agric. Food. Chem, 51, 609-614. [19] Zakirov SK, Kasymo SZ, Sidyakin GP. (1975). Sesquiterpene lactones from Jurinea maxima. Khim. Prir. Soedin. 5, 656-657. http://ijlssr.com IJLSSR © 2015 All rights are reserved