Asian Pacific Journal of Tropical Medicine (2014)9-15 9

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Document heading doi: , lipid peroxidation inhibition and free radical scavenging efficacy of a diterpenoid compound sugiol isolated from Metasequoia glyptostroboides

1 1 Vivek K. Bajpai , Ajay Sharma , Sun Chul Kang2*, Kwang-Hyun Baek1* 1School of Biotechnology, Yeungnam University, Gyeongsan, Gyeongbook 712-749, Republic of Korea 2Department of Biotechnology, Daegu University, Gyeongsan, Gyeongbook 712-714, Republic of Korea

ARTICLE INFO ABSTRACT

Article history: Objective: To investigateMetasequoia the antioxidant glyptostroboides efficacy of M.a biologically glyptostroboides active diterpenoid compound ( ) Received 10 September 2013 sugiol isolatedMethods: from in various antioxidantM. Received in revised form 15 November 2013 models.glyptostroboides An type diterpenoid sugiol, isolated from ethyl acetate extract of Accepted 15 December 2013 cones, was analyzed for its antioxidant efficacy as reducing power ability and Available online 20 January 2014 lipid peroxidation inhibition as well as its ability to scavenge free radicalsResults: such as 1,1-diphenyl- 2-picryl hydrazyl, nitric oxide, superoxide and hydroxyl radicals. The sugiol showed significant and concentration-dependent antioxidant and free radical scavenging activities. Keywords: Consequently, the sugiol exerted lipid peroxidation inhibitory effect by 76.5% as compared Metasequoia glyptostroboides to 毩-tocopherol (80.13%) and butylated hydroxyanisole (76.59%). In addition, the sugiol had significant scavenging activities of 1,1-diphenyl-2-picryl hydrazyl, nitric oxide, superoxide Sugiol and hydroxyl free radicals in a concentration-dependent manner by 78.83%, 72.42%, 72.99% Diterpenoid and 85.04%, when compared to the standard compound ascorbic acid (81.69%, 74.62%, 73.00% Antioxidant 73 79%) (84 09% 78 61% 74 45% 70 02%) and . and Conclusions: 毩-tocopherol/butylated hydroxyanisole . , . , . and . M., Free radicals glyptostroboidesrespectively. These findings justify the biological and traditional uses of Lipid peroxidation or its secondary metabolites as confirmed by its promising antioxidant efficacy.

2 (H2O2) and singlet oxygen (O .) are the by-products of 1. Introduction cellular metabolism with most biological significance[1]. The ROS are potentially damaging transient chemical species C urrent life style causes overproduction of free radicals which are produced by a number of exogenousγ sources such and (ROS). Free radicals are as ionizing radiations (ultraviolet rays and -rays), tobacco generated through normal reactions within the body smoke, pesticides, pollutants, and some medications. Such during respiration in aerobic organisms which can exert species are considered to be important causative factors in diverse functions like signaling roles and provide defense decreasing cell fluidity, permeability, cell dysfunction and against infections[1]. The ROS such as nitric oxide radical, mutations[2]. Many degenerative human diseases including superoxide radical, hydroxyl radical, hydrogen peroxide cancer, cardio- and cerebro-vascular diseases have been recognized as a possible consequence of free radical

*Corresponding author: Prof. Kwang-Hyun Baek, School of Biotechnology, Yeungnam damage to lipids, proteins and nucleic acids[2]. Natural University, Gyeongsan, Gyeongbook 712-749, Republic of Korea. Tel: +82-53-810-4769 protect the living system from oxidative stress Fax: +82-53-810-4769 and associated diseases and therefore play an important E-mail: [email protected] [3] Prof. Sung Chul Kang, Department of Biotechnology, Daegu University, Gyeongsan, role in health care system . Gyeongbook 712-714, Republic of Korea. Food industry has long been concerned with issues Tel: +82-53-850-6553 [4] Fax: +82-53-850-6554 such as rancidity and oxidative spoilage of foodstuffs . E-mail: [email protected] T Foundation project: This research was supported by a grant (NRF-2011-0008199) he enzymatic oxidation as well as auto-oxidation of from the Basic Science Research Program through the National Research Foundation lipids during storage and processing is the major reaction of Korea. Vivek K. Bajpai et al./Asian Pacific Journal of Tropical Medicine (2014)9-15 10 responsible for the deterioration in food quality affecting brain extract, nitro blue tetrazolium (NBT), ferric chloride, color, flavor, texture and nutritive value of foods[2]. potassium ferricyanide and gallic acid as well as standard Antioxidants are often added to foods to prevent the radical antioxidant compound ascorbic acid, BHA and 毩 chain reactions of oxidation by inhibiting the initiation and -tocopherol were purchased from Sigma-Aldrich (St. Louis, propagation steps leading to the termination of the reaction USA) and were of analytical grade. Spectrophotometric and a delay in the oxidation process. measurements were done using a 96-well micro-plate Synthetic phenolic antioxidants such as butylated enzyme linked immunosorbent assay (ELISA) reader (Infinite hydroxytoluene (BHT), butylated hydroxyanisole (BHA) M200, Teacan, Mannedorf, Switzerland). and tert-butylhydroxyquinone (TBHQ) effectively inhibit 2.2. Plant material lipid oxidation. However, these commonly used synthetic antioxidants are restricted by legislative rules, because M. glyptostroboides they are suspected to have toxic effects and are considered The male cones of were collected carcinogenic by nature[4]. Therefore, there has been a locally from Pohang city, Republic of Korea, in November considerable interest by the food industry and a growing and December 2008 and identified by the morphological trend in consumer preferences for using natural antioxidants features and the database present in the library at the over synthetic compounds in order to eliminate synthetic Department of Biotechnology, Daegu University, Korea. The antioxidants in food applications, giving more impetus to cones were dried under shade at room temperature without explore natural sources of antioxidants. This has encouraged exposure of sunlight. A voucher specimen (DUB-0038) both food scientists and health professionals to work on was deposited in the herbarium of College of Engineering, antioxidant molecules from natural origin. Nowadays, there Department of Biotechnology, Daegu University, Korea. has been a convergence of interest among researchers to 2.3. Extraction and isolation of sugiol find out the role of natural antioxidants in the diet, and their impact on human health has come under huge attention[5]. Metasequoia glyptostroboides M. M. glyptostroboides Miki ex Hu ( Dried cones of (2 kg) were milled glyptostroboides ) is a deciduous coniferous tree of the into powder and then extracted with ethyl acetate at room redwood family, . This species of the genus temperature for 12 days. The extract was evaporated under Metasequoia has been propagated and distributed in many reduced pressure using a rotary evaporator (EYELA N1000, parts of Eastern Asia and North America as well as in Japan). The dried ethyl acetate extract (7 g) was subjected Europe. Previously, we reported various biological properties to column chromatography over silica gel (230-400 meshes, M. glyptostroboides of various essential oils derived from Merck, Darmstadt, Germany) and was eluted with hexane- such as antibacterial[6,7], antioxidant/antibacterial[8], ethyl acetate-methanol solvent system to give 20 fractions. antidermatophytic[9] and antifungal[10] activities. In addition, Of the fractions obtained, fraction-14 was further purified by M. the antibacterial activities of terpenoid compounds from preparative thin layer chromatography over silica gel GF254 glyptostroboides have also been reported against foodborne using hexane-ethyl acetate (2:1, v:v) as a mobile phase to pathogenic bacteria[11]. give one compound (122 mg) which was characterized as M. glyptostroboides The biological efficacy of has been sugiol on the basis of spectral data analysis[12]. in vitro in vivo reported previously both and , however, no 2.4. Determination of DPPH radical scavenging assay research has been reported on the antioxidant and free M. glyptostroboides radical scavenging efficacy of sugiol from including its lipid peroxidation inhibitory effect. Hence, in The antioxidant activity of sugiol, based on the scavenging addition to our continuous efforts to investigate biologically of stable DPPH free radical, was determined by the method M. glyptostroboides active secondary metabolites from , described previously with a minor modification[13]. Different in this study, we assayed the antioxidant and free radical concentrations of sugiol and reference compounds such as scavenging efficacy of sugiol, an abietane type diterpenoid ascorbic acid and 毩-tocopherol (25-150 毺g/mL) were added compound in various antioxidant models. to 0.004% methanolic solution of DPPH (1:1, v:v) in a 96-well microplate. The mixture was incubated at 37 曟 in dark for 30 min with shaking at 150 r/min. Absorbance was recorded 2. Materials and methods at 517 nm using the 96-well ELISA reader against a blank sample. All the tests were run in triplicate. Ascorbic acid 2.1. Chemicals and instrument and 毩-tocopherol were used as reference compounds. The percent inhibition activity was calculated using the formula: A A A × The chemicals and reagents used in this study such as 1,1- Percent inhibition (%) = ( control- test)/ control 100. (1) A diphenyl-2-picryl hydrazyl (DPPH), sodium nitroprusside where, control is the absorbance of the control reaction at 517 nm A (SNP), Griess reagent, trichloroacetic acid (TCA), bovine and test represents the absorbance of a test reaction at 517 nm. Vivek K. Bajpai et al./Asian Pacific Journal of Tropical Medicine (2014)9-15 11 2.5. Determination of nitric oxide (NO) radical scavenging quantification of the degradation product of 2-deoxy-2- assay ribose sugar by condensation with 2-thiobarbituric acid (TBA) H F . ydroxyl radicals3+ were generated by the enton In aqueous solution at physiological pH, SNP automatically reaction using Fe -ascorbate-EDTA-H2O2 system. The generates NO, which intermingles with oxygen to generate reaction mixture in a final volume of 240 毺L contained nitrite ions that can be anticipated by the Griess reagent 2-deoxy-2-ribose (3 mmol/L), KH2PO4-KOH buffer (20 mmol/L, (10 g/L sulphanilamide, 20 mL/L phosphoric acid and 1 g/L pH 7.4), FeCl3 (0.1 mmol/L), ethylenediamine tetra-acetic naphthyl ethylene diamine dihydrochloride)[14]. Scavengers acid (EDTA) (0.1 mmol/L), H2O2 (2 mmol/L), ascorbic acid of free radicals result in the reduced production of NO. In (0.1 mmol/L) and various concentrations (100-500 毺g/mL) of this assay, the solution of SNP (10 mmol/L) in phosphate sugiol or standard compounds. After incubation for 45 min buffer saline (PBS, pH 7.4) was mixed with different at 37 曟, 40 毺L of 2.8% (v:v) TCA, and 40 毺L of TBA [0.5% concentrations of sugiol, ascorbic acid and 毩-tocopherol (20- (v:v) in 0.025 mol/L NaOH solution containing 0.2 g/L BHA] 100 毺g/mL). The mixture was incubated at 37 曟 for 60 min were added in the reaction mixture, and the mixture was in light. The half quantity of aliquots was taken and mixed incubated at 95 曟 for 15 min to develop the pink color. After with equal quantity of the Griess reagent, and the mixture cooling, the absorbance was measured at 532 nm against was incubated at 25 曟 for 30 min in dark. The absorbance an appropriate blank solution. All tests in this assay were of pink chromophore generated during diazotization of performed three times. Ascorbic acid and BHA were used nitric ions with sulphanilamide and subsequent coupling as positive controls. The percent inhibition activity was calculated using the formula: with naphthyl ethylene diamine dihydrochloride was read A A A × 546 [14] A Percent inhibition (%) = ( control- test)/ control 100. (4) at nm against a blank . ll the tests were performed A A where, control is the absorbance of the control reaction at in triplicate. scorbic acid and 毩-tocopherol were used A as standard reference compounds. The percent inhibition 532 nm and test represents the absorbance of a test reaction activity was calculated using the formula: at 532 nm. A A A × Percent inhibition (%) = ( control- test)/ control 100. (2) A 2.8. Lipid peroxidation assay where, control is the absorbance of the control reaction at A 546 test nm and represents the absorbance of a test reaction 3+ at 546 nm. The Fe /ascorbic acid dependent non-enzymatic lipid peroxidation in bovine brain extract was performed 2.6. Determination of superoxide radical scavenging assay according to the previous method with minor modifications[17]. The reaction mixture, in the absence and presence of sugiol Superoxide radical scavenging activity of sugiol was or reference compounds (50-250 毺g/mL), containing 50 毺L of measured by the reduction of NBT according to the 5 mg/mL bovine brain phospholipids, 1 mmol/L FeCl3 and 1 previously reported method with minor modifications[15]. mmol/L ascorbic acid in 20 mmol/L phosphate buffer with a In this assay, the non-enzymatic phenazine methosulfate- final volume of 330 毺L, was incubated at 37 曟 for 1 h. The nicotinamide adenine dinucleotide (PMS/NADH) system hydroxyl radicals generated in the reaction initiated the lipid generates superoxide radicals, which reduce NBT to a purple peroxidation, resulting in malondialdehyde (MDA) production color formazan. The reaction mixture (150 毺L) contained that was measured by TBA reaction using an ELISA reader. phosphate buffer (0.2 mol/L, pH 7.4), NADH (73 毺mol/L), All tests in this assay were performed three times. BHA and NBT (50 毺mol/L), PMS (15 毺mol/L) and various 毩-tocopherol were used as positive controls. The percent (50 250 L) A inhibition activity was calculated using the formula: concentrations - 毺g/m of the sugiol solution. fter A A A × 60 Percent inhibition (%) = ( control- test)/ control 100. (5) incubation for min at room temperature, the absorbance A 560 where, control is the absorbance of the control reaction at of the reaction mixture was measured at nm against an A appropriate blank to determine the quantity of formazan 532 nm and test represents the absorbance of a test reaction generated. All tests were performed three times. Ascorbic at 532 nm. T acid and 毩-tocopherol were used as positive controls. he 2.9. Reducing power assay percent inhibition activity was calculated using the formula: A A A × (%) ( control test) control ( ) Percent inhibition = - / 100. 3 + A 3 where, control is the absorbance of the control reaction at The ferric ion (Fe ) reducing power of the sugiol was A 560 nm and test represents the absorbance of a test reaction determined by the method described previously with minor at 560 nm. modifications[18]. Aliquots (50 毺L) of different concentrations of sugiol (5-25 g/mL) were mixed with 50 L phosphate 2.7. Determination of hydroxyl radical scavenging assay 毺 毺 buffer (0.2 mol/L, pH 6.6) and 50 毺L potassium ferricyanide (10 g/L), followed by incubation at 50 曟 for 20 min in dark. A previously described method was adopted for After incubation, 50 毺L of TCA (10%, v:v) was added to determining the hydroxyl radical scavenging activity of terminate the reaction and the mixture was subjected to sugiol with minor modifications[16]. The assay is based on centrifugation at 3 000 r/min for 10 min. For final reaction Vivek K. Bajpai et al./Asian Pacific Journal of Tropical Medicine (2014)9-15 12 mixture, the supernatant (50 毺L) was mixed with 50 毺L 100 Sugiol Ascorbic acid 毩-tocopherol 3 ( ) distilled water and 10 毺L FeCl solution 1 g/L . The reaction a b a mixture was incubated for 10 min at room temperature 80 c c d 700 and the absorbance was measured at nm against an e e e 60 appropriate blank solution. A higher absorbance of the f g fg A h i hi reaction mixture indicated greater reducing power ability. ll 40 tests were run in triplicate. Ascorbic acid and 毩-tocopherol Percentage inhibition (%) Percentage were used as positive controls. 20

2.10. Statistical analysis 0 25 50 75 100 150 Concentration (毺g/mL) ± A SD Figure 1. ll data are expressed as the mean by measuring three DPPH radical scavenging activity of a diterpenoid sugiol independent replicates. Analysis of variance using one-way M. glyptostroboides ’ from and standard antioxidants, ascorbic acid and ANOVA followed by Duncan s test was performed to test the 毩-tocopherol. Different superscripts in eachP column indicate the significant significance of differences between means obtained among differences between the means ( <0.05). the treatments at the 5% level of significance using the SAS software (Version SAS 9.1; SAS Institute Inc., Cary, NC, USA). 3.3. NO radical scavenging activity

As shown in Figure 2, both sugiol and positive controls 3. Results P showed significant NO radical scavenging activity ( <0.05) 3.1. Identification of sugiol in a concentration-dependent manner. In this assay, sugiol caused a concentration-dependent inhibition of NO, and ( %) M. glyptostroboides the highest inhibitory effect 72.42 was observed at the The ethyl acetate extract of cones concentration of 100 毺g/mL. On the other hand, ascorbic after column chromatography over silica gel yielded a pure acid and 毩-tocopherol as positive controls (100 毺g/mL) had compound, which was obtained as yellow glass crystal with about 74.62% and 78.61% of inhibitory effect on scavenging a specific melting point (282-284 曟). The 1H NMR data of NO radical, respectively. (pyridine-d5, 250 MHz) showing two singlet protons at d 8 39 7 16 ( 1H ) . and . each , s , an aliphatic methine signal at d 100 Sugiol Ascorbic acid 毩-tocopherol 3.59 (1H, m, signals partially overlapped), and five terminal J J a ( ) ( 80 b b methyl groups at d 1.35 3H, d, = 6.8 Hz , 1.33 3H, d, = c 6.8 Hz), 1.11 (3H, s, Me-20), 0.83 (3H, s, Me-19), 0.79 (3H, s, d d 60 e e e Me-18), as well as the 13C NMR data displaying 20 carbon f f f 197 6 signals including a carbonyl group at d . strongly 40 g g g

suggested that this compound should be an abietane inhibition (%) Percentage diterpenoid. The structure of this compound was determined 20 to be sugiol by 1D and 2D NMR analysis and also confirmed 0 by comparing the physical and spectroscopic data with those 20 40 60 80 100 in the literature[12,19]. Concentration (毺g/mL) Figure 2. 3.2. DPPH radical scavenging activity NitricM. glyptostroboides oxide radical scavenging activity of a diterpenoid sugiol from and standard antioxidants, ascorbic acid and 毩-tocopherol. F 1 DPPH Different superscripts in eachP column indicate the significant igure shows the percentage of radical scavenging differences between the means ( <0.05). capacity of sugiol in comparison with ascorbic acid and 毩-tocopherol as reference compounds. A concentration- 3.4. Superoxide anion radical scavenging activity dependent response relationship was found in the DPPH radical scavenging capacity and the activity was increased The effect of the sugiol on superoxide radical was with the increase of sample concentration. The sugiol determined by the PMS-NADH superoxide generating showed maximum 78.83% inhibition of DPPH radicals, system and the results are shown in Figure 3. All the while ascorbic acid and 毩-tocopherol showed about tested samples significantly scavenged the superoxide P 81.69% and 84.09% inhibitory effect, respectively within ( ) P radicals in a concentration-dependent manner <0.05 . the concentration range. Significant ( <0.05) scavenging of It has been reported that antioxidant properties of some DPPH radicals was evident at all the tested concentrations of phenolic compounds are effective mainly via scavenging of sugiol and reference compounds (25-150 毺g/mL). superoxide anion radicals[4]. In this assay, addition of sugiol Vivek K. Bajpai et al./Asian Pacific Journal of Tropical Medicine (2014)9-15 13 3+ and standard compounds (ascorbic acid and 毩-tocopherol) at Fe and ascorbate which attack the biological material. This the concentration of 250 毺g/mL showed 72.99%, 73.00% and leads to the formation of MDA and other aldehydes, which 74.45% superoxide radical scavenging effect, respectively. form a pink chromogen with TBA, absorbing at 532 nm. As shown in Figure 5, the sugiol demonstrated considerable 100 Sugiol Ascorbic acid 毩-tocopherol amount of lipid peroxidation inhibitory effect by 76.50%, while -tocopherol and BHA significantly inhibited lipid 80 a a a 毩 % % b b b peroxidation by 80.13 and 76.59 , respectively. The results 60 c were concentration-dependent and considered statistically d d P e e e significant ( <0.05). 40 g f f Percentage inhibition (%) Percentage 20 100 Sugiol 毩-tocopherol BHA a b b 0 80 50 100 150 200 250 c c c Concentration ( g/mL) 毺 60 d d d Figure 3. e e e SuperoxideM. glyptostroboides radical scavenging activity of a diterpenoid 40 sugiol from and standard antioxidants, ascorbic f f f acid and -tocopherol. inhibition (%) Percentage 毩 20 Different superscripts in eachP column indicate the significant differences between the means ( <0.05). 0 3.5. Hydroxyl radical scavenging activity 50 100 150 200 250 Concentration (毺g/mL) Figure 5. M. glyptostroboides Lipid peroxidation inhibitory effect of a diterpenoid sugiol The scavenging effect of sugiol against hydroxyl radicals from and standard antioxidants, 毩-tocopherol and BHA. was investigated using the Fenton reaction. As presented in D ifferent superscripts in eachP column indicate the significant Figure 4, the percentage inhibition of sugiol, ascorbic acid differences between the means ( <0.05). and BHA on hydroxyl radical scavenging was found to be 85.04%, 73.79%, 70.02%, respectively. The results showed 3.7. Reducing power activity significant antioxidant activity in a concentration-dependent P 3+ 2+ manner ( <0.05). The ability of the sugiol to quench hydroxyl As illustrated in Figure 6, the conversion of Fe to Fe in radicals seems to be directly related to the prevention of the presence of sugiol and reference compounds could be propagation of lipid peroxidation; because sugiol seems to measured as their reductive ability. At the concentration of be a good scavenger of active oxygen species, it will thus 25 L 毺αg/m , the absorbance values of sugiol, ascorbic acid reduce the rate of the chain reaction. and -tocopherol were measured to be 1.09, 1.21, and 1.14, respectively. The results showed a concentration-dependent 100 Sugiol Ascorbic acid BHA P a significant increase ( <0.05) in reductive ability of the test b 80 c d samples. These results demonstrated that sugiol had marked e e 3+ f ferric ions (Fe ) reducing ability and had electron donor g g 60 h properties for neutralizing free radicals by forming stable i i 40 products. j k k Percentage inhibition (%) Percentage 1.4 Sugiol Ascorbic acid 毩-tocopherol 20 a b b 1.2 c bc 0 d 100 200 300 400 500 1.0 ef e f g Concentration (毺g/mL) 0.8 h hi i Figure 4. 0.6 M. glyptostroboides Hydroxyl radical scavenging activity of a diterpenoid sugiol j j from and standard antioxidants, ascorbic acid and 0.4 Absorbance at 700 nm Absorbance BHA. 0.2 Different superscripts in eachP column indicate the significant differences between the means ( <0.05). 0 5 10 15 20 25 3.6. Lipid peroxidation inhibitory activity Concentration (毺g/mL) Figure 6. M. glyptostroboides Reducing power activity of a diterpenoid sugiol from and standard antioxidants, ascorbic acid and I n this assay, the peroxidation of bovine brain phospholipid 毩-tocopherol. D extract was induced by ferric chloride and ascorbic acid as ifferent superscripts in eachP column indicate the significant reducing agents. Hydroxyl radicals are generated by mixing differences between the means ( <0.05). Vivek K. Bajpai et al./Asian Pacific Journal of Tropical Medicine (2014)9-15 14 4. Discussion radical scavenging capacity of sampleset isal directly related to its antioxidant activity[32]. Hagerman . explained that The scavenging activity on DPPH radicals assay is high molecular weight phenolic compounds comprising generally used as a basic screening method for testing the hydroxyl group and aromatic ring serve as potent free radical anti-radical activity of a large variety of compounds[20]. scavengers[33]. Hydroxyl radical scavenging efficacy of This assay is based on the measurement of the ability of diterpenoid compounds has been confirmed previously[23,24]. antioxidants to scavenge the stable radical DPPH, widely Several factors to food commodities lead to the quality used in evaluating the antioxidant activities in a relatively deterioration. Among these, an undesirable factor, i.e., lipid short time as compared to the other methods. In this assay, auto-oxidation, is one of the most concerned. The need of the color of DPPH radical changes from violet to yellow protecting food against oxidative degradation has prompted upon reduction which is demonstrated by the decrease in the wide usage of food additives from natural origin. Lipid absorbance at 517 nm. The free DPPH radical is reduced to peroxidation leads to rapid development of rancid and stale the corresponding hydrazine when it reacts with hydrogen flavors, and it is considered as a primary mechanism of F donors. or being an easy and accurate method, it has been quality deterioration in lipid foods[34]. Synthetic antioxidants, recommended to measure the antioxidant activity of samples e.g., BHA, are added in food during processing to suppress of different origin[21]. Free radical scavenging is one of lipid peroxidation and consequently to improve food quality the recognized mechanisms by which antioxidants inhibit and stability. The phenolic compounds and other chemical lipid peroxidation[22]. DPPH radical scavenging activity of diterpenoid compounds has been confirmed previously[23,24]. components may suppress lipid peroxidation through NO is a free radical with a single unpaired electron. different chemical mechanisms, including free radical NO L NO [25] I quenching, electron transfer, radical addition or radical is formed from -arginine by synthase . t can [35] also be formed from the reaction of peroxyl radical and recombination . In addition, lipid peroxidation of cell NO, polluted air, and smoking[26]. NO itself is not a very membrane is associated with various pathological events [36] reactive free radical, however, overproduction of NO is such as atherosclerosis, inflammation and liver injury . involved in ischemia reperfusion, neurodegenerative Previous reports have confirmed the lipid peroxidation [23,24] and chronic inflammatory diseases like rheumatoid inhibitory effect of various terpenoid compounds . arthritis. Nitric dioxide adds to double bonds and extracts The reducing property of test compounds indicates they can be used as electron donors which reduce the oxidized labile hydrogen atoms initiating lipidet peroxidation al and production of free radicals. Marcocci . reported that intermediates of lipid peroxidation processes, therefore, NO scavengers compete with oxygen, resulting in a lower they can act as primary and secondary antioxidants. In the [27] ( ) production of NO . The metabolite ONOO- peroxynitrite present study, 3+assay of reducing power was2+ based on the F F is extremely reactive, directly inducing toxic reactions, reduction of e /ferricyanide complex to e in the presence2+ SH ( ) T F including -group oxidation, protein tyrosine nitration, of reductants antioxidants in the tested samples. he e’ lipid peroxidation, and DNA modifications[28]. NO radical was then monitored by measuring the formation of Perl s scavenging activity of terpenoid compounds has been Prussian blue at 700 nm[18]. The increased reducing ability confirmed previously[23,24]. observed may be due to the formation of reductants which Superoxide anion, which is a reduced form of molecular could react with free radicals to stabilize and terminate oxygen, is an initial free radical formed from mitochondrial radical chain reactions during fermentation, converting electron transport systems. Superoxide anions serve as them to more stable products[37]. Some of the terpenoid precursors to active free radicals that have the potential to compounds have been found to be potent reductants[23,24]. react with biological macromolecules and thereby induce On the basis of the results obtained in the present study, [29] tissue damage . Superoxide has also been observed to it can be concluded that sugiol exhibited potent antioxidant directly initiate lipid peroxidation and plays an important and free radical scavenging activities. Moreover, the ROS role in the formation of other like hydroxyl radicals, hydrogen donating ability of sugiol has been proven through which induce oxidative damage in lipids, proteins, and the assessment of reducing power ability and radical DNA. Previously diterpenod compounds have been found scavenging activities. Sugiol also exerted significant lipid to possess significant superoxide radical scavenging peroxidation inhibitory activity of considerable interest. activity[23,24]. These results confirm the efficacy of sugiol as a significant Hydroxyl radicals are extremely reactive free radicals formed in biological systems and have been implicated as a source of natural antioxidant to stabilize the foods from highly damaging species in free radical pathology, capable oxidative deterioration and to prevent the progress of variousin vivo of damaging almost every molecule found in living cells. oxidative stress-induced diseases. However, the These radicals can be formed from a superoxide anion and safety needs to be thoroughly investigated in experimental hydrogen peroxide in the presence of copper or iron ions[30]. rodent models prior to its practical application. Hydroxyl radicals are very strongly reactive oxygen species, and there is no specific enzyme to defend against them in Conflict of interest statement human[31]. Therefore, it is important to discover natural compounds with good scavenging capacity against these reactive oxygen species. It is well established that hydroxyl We declare that we have no conflict of interest. Vivek K. Bajpai et al./Asian Pacific Journal of Tropical Medicine (2014)9-15 15 Acknowledgments Nigella sativa and derived thymoquinone inhibit eicosanoid Planta generation in leukocytes and membrane lipid peroxidation. Med 61 This research was supported by a grant (NRF-2011- 1995; : 33-36. ) [18]O yaizu M. Studies on product of browning reaction prepared from 0008199 from the Basic Science Research Program through Jpn J Nutri 44 the National Research Foundation of Korea. glucose amine. 1986; : 307-315. [19]C hang HS, Cheng KP, Choang TF, Chow HF, Chui KY, Hon PM, et al. Structure elucidation and total synthesis of new tanshinones References J Org Chem isolated from Bunge (Danshen). 55 1990; : 3537-3543. Food Chem [1] H ancock JT, Desikan R, Neill SJ. Role of reactive oxygen species in [20]S harma OP, Bhat TK. DPPH antioxidant assay revisited. 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