Isolation and Antioxidant Activity Evaluation of Two ... - Springer Link

Biotechnology and Bioprocess Engineering 17: 1031-1040 (2012) DOI 10.1007/s12257-012-0115-1

RESEARCH PAPER

Isolation and Antioxidant Activity Evaluation of Two New Phthalate Derivatives from Seahorse, Hippocampus Kuda Bleeler Zhong-Ji Qian, Kyong-Hwa Kang, and Se-Kwon Kim

Received: 20 February 2012 / Revised: 19 May 2012 / Accepted: 20 May 2012 © The Korean Society for Biotechnology and Bioengineering and Springer 2012

Abstract Seahorse (Hippocampus Kuda Bleeler) has been used as traditional medicine for thousands of years, in Eastern Asia. In this study of the methanol extract of fresh Hippocampus Kuda, the new compounds 2-ethyldecyl 2ethylundecyl phthalate (1), 2, 12-diethyl-11-methylhexadecyl 2-ethyl-11-methylhexadecylphthalate (2), along with a known Bis(2-ethylheptyl) phthalate (3) were isolated. They were tested for their antioxidant activities, including lipid peroxidation inhibitory activity, DPPH radical scavenging, hydroxyl radical scavenging, superoxide anion radical scavenging, alkyl radical scavenging, and cellular radicals; these can be detected using a fluorescence probe, 2’,7’dichlorofluorescin diacetate (DCFH-DA), which could be converted to highly fluorescent dichlorofluorescein (DCF) with the presence of intracellular ROS on mouse macrophages, RAW264.7 cell. Compound (2) exhibited the highest antioxidant activity and inhibitory intracellular ROS than another compounds (1, 3). Furthermore, MTT assay showed no cytotoxicity on mouse macrophages cell (RAW264.7) and human fetal lung fibroblast cell line (MRC-5). This antioxidant property depends on concentration and increasing with increased amount of the compound. Keywords: hippocampus kuda bleeler, phthalate, antioxidant activity, free radical scavenging, reactive oxygen species (ROS)

Zhong-Ji Qian Department of Marine Life Science and Marine Life Research and Education Center, Chosun University, Gwangju 501-759, Korea Kyong-Hwa Kang, Se-Kwon Kim* Marine Bioprocess Research Center, Pukyong National University, Busan 608-737, Korea Tel: +82-51-629-7094; Fax: +82-51-629-7099 E-mail: [email protected]

1. Introduction Traditional medicine came into existence long before Western medicine was developed in Europe. Recently, the potential of natural products developed from organisms, and especially from marine medicinal organism used in traditional medicine has been recognized by the scientific community in the Western world. Seahorse, a marine teleost fish, is well known not only for its special medicinal composition, but also for its unusual features including male pregnancy. Although seahorses are usually treated as pets, seahorse (Hippocampus kuda Bleeker) is used as one of the most famous and expensive materials of traditional Chinese medicine (TCM). At present, the natural source of seahorse has been dramatically reduced as a result of over fishing. Therefore, it is urgent to learn more about this unique creature physiologically and pharmacologically in order to efficiently protect and properly harness this precious species. Seahorse belongs to the Syngnathidae of Syngnathiformes, in Steichthyes of the vertebrate phylum. Most animals in Syngnathidae can be used as TCM material and were well documented by all versions of the China Pharmacopoeia, even as early as in the Liang Dynasty (A.D. 502 ~ 557). According to the Yin Yang theory described in TCM books, seahorse has the effect on tonifying kidney and activating Yang. The former function is essentially related to the regulation of urinogenital, reproductive, nervous, endocrine, and immune systems; the latter is referred to enhance male sexual function. Recent, pharmacological studies suggested that seahorse, not only had hormone-like activities [1], boosting hematopoiesis and has shown activities of antioxidant, anti-tumor, antifatigue, and Ca2+ channel blocking [2-5]. There has been considerable interest among basic and clinical researchers in traditional medicines in recent years,

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Biotechnology and Bioprocess Engineering 17: 1031-1040 (2012)

for their long history of clinical practice and reliable therapeutic efficacy. However, the components of TCM are complex mixtures, almost consisting of hundreds or even thousands of different compounds; however, on a few compounds are responsible for the pharmaceutical and/or toxic effects. Furthermore, the effective components in TCM have been a great challenge for many investigators. The conventional procedure for finding bioactive components is to extract the effective compound groups or purification compounds in TCM with a method of phytochemistry, followed by animal pharmacological experiments. However, this method is time-consuming, arduous, expensive, and of low efficiency for direct screening of bioactive compounds from TCM. Previous studies on the active components of seahorse were carried out by various methods of medicinal chemistry, which focused on analyses of trace elements, amino acids, and soluble components in organic reagents, such as fatty acids, phospholipids, and steroids. However, there were a few reports on seahorse in natural products, and the isolation of physiologically and pharmacologically bioactive compounds. Cardiovascular disease, cancer, and age-related disorders claim millions of lives each year and their prevention and treatment are very important objectives for modern medical research. Oxidative stress refers to the undue oxidation of bio-molecules leading to cellular damage. Oxidative stress is widely recognized as a factor in the onset and progression of various diseases, such as cancer, arteriosclerosis, and neural degenerative disorders. The reactive oxygen species (ROS) are known to play a major role in either the initiation or progression of carcinogenesis by inducing oxidative stress [4,6]. In this study, we report the isolation and structure elucidation of antioxidant activity compounds (two new compounds) for the first time from Seahorse, Hippocampus Kuda Bleeler.

1640 Medium, trypsin-EDTA, penicillin/streptomycin/amphotericin (10,000 U/mL, 10,000 µg/mL, and 2,500 µg/mL, respectively) and fetal bovine serum (FBS) were obtained from Gibco BRL, Life Technologies (USA). MTT (3-(4,5dimethyl-2-yl)-2,5-diphenyltetrazolium bromide) reagent, phoroglucinol, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), α-(4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN), 2,2'azobis(2-amidinopropane) dihydrochloride (AAPH) and myeloperoxidase (MPO) reagent were purchased from Sigma Chemical Co. (St. Louis, MO, USA). 2’,7’-dichlorofluorescin diacetate (DCFH-DA) and monobromobimane were purchased from Molecular probes (Eugene, OR, USA). Other chemicals and reagents used were of analytical grade and commercially available.

2. Materials and Methods 2.1. Materials The live adults of sea horse were collected from Zhoushan Island, Zhejiang, China, in October, 2005, and freeze-dried after removing the internal organs. The seahorses were identified as Hippocampus Kuda Bleeler by Professor Ming-Lu Deng (Zoologist, Changchun University of Chinese Medicine, China). Murine macrophage (RAW264.7), human fetal lung fibroblast (MRC-5) and human promyelocytic leukemia (HL60) cell lines were obtained from American Type of Culture Collection (Manassas, VA, USA). Dulbecco’s Modified Eagle’s Medium (DMEM), RPMI

2.2. Extraction and isolation The freeze-dried H. Kuda Bleeler (2 kg) was ground into powder and extractedd at room temperature with MeOH (15 L) for 15day, and the solvent was evaporated to give a crude MeOH extract. The methanol extract was subjected to silica gel flash chromatography and eluted with nhexane/EtOAc/MeOH. Fraction 5 (n-hexane/EtoAc, 5:1) was chromatographed over ODS by elution with MeOH/ H2O to yield compounds 1 ~ 3, respectively. The isolates were further purified by HPLC (Dionex, ODS, MeOH) utilizing a 40 min gradient program of 50 to 100% MeOH in H2O to furnish compound 1 (21.5 mg), compound 2 (15.6 mg), and compound 3 (17.8 mg). 2.3. Structural confirmation of compounds After isolation, structure of compounds were determined using FT-IR spectra were recorded on a IFS-88 spectrometer (Bruker, USA), proton NMR (1H NMR, 400 MHz) and carbon NMR (13C NMR, 100 MHz) in a CDCl3 environment on a JNM-ECP-400 spectrometer (JEOL, Japan), using TMS or solvent peaks as reference standard. Optical rotations were determined on an Elmer model 341 polarimeter (Perkin, USA). MS spectra were obtained on JMS-700 spectrometer (JEOL, Japan). UV/visible spectra were measured on a U-2001 specromater (Hitach, Japan). CD spectra were taken on a J-715 spectropolarimeter (JASCO, Japan). 2.4. Lipid peroxidation inhibition assay The total antioxidative activity was measured in a linoleic acid model system according to the methods of Osawa and Namiki [7]. Briefly, a sample (1.3 mg) was dissolved in ethanol, and added to a solution of 0.13 mL of linoleic acid and 10 mL of 99.5% ethanol. Then the total volume was adjusted to 25 mL by adding distilled water. The mixture was incubated in a conical flask with a screw cap at 40 ±

Isolation and Antioxidant Activity Evaluation of Two New Phthalate Derivatives from Seahorse, Hippocampus Kuda Bleeler

1oC in a dark room and the degree of oxidation was evaluated by measuring the ferric thiocyanate values. The reaction solution (100 µL) was incubated in the linoleic acid model system and was mixed with 4.7 mL of 75% ethanol, 0.1 mL of 30% ammonium thiocyanate, and 0.1 mL of 2 × 10-2 M ferrous chloride solution in 3.5% HCl. After 3 = min, the thiocyanate value was measured by reading the absorbance at 500 nm following color development with FeCl2 and thiocyanate at different intervals during the incubation period at 40 ± 1oC. 2.5. Measurement of free radicals scavenging activity by electron spin resonance (ESR) spectrometer Different radicals were generated according to previously mentioned procedures and spin adducts were recorded using a JES-FA electron spin resonance (ESR) spectrometer (JEOL Lts., Tokyo, Japan). Radical scavenging ability was calculated using the following equation in which H and H0 were relative peak height of radical signals with and without sample, respectively. 1 – H- × 100% Radical scavening activity = ---------H0

2.6. DPPH radical scavenging activity DPPH radical scavenging activity was measured using the method described by Nanjo et al. [8]. A 30 µL compounds solution (or ethanol itself as control) was added to 30 µL of DPPH (60 µM) in ethanol solution. After mixing vigorously for 10 sec, the solution was then transferred into a 100 µL quartz capillary tube, and the scavenging activity of peptide on DPPH radical was measured using a JES-FA ESR spectrometer (JEOL Ltd., Tokyo, Japan). The spin adduct was measured on an ESR spectrometer exactly 2 min later. Experimental conditions as follows: magnetic field, 336.5 ± 5 mT; power, 5 mW; modulation frequency, 9.41 GHz; amplitude, 1 ×1,000; sweep time, 30 sec. 2.7. Hydroxyl radical scavenging activity Hydroxyl radicals were generated by iron-catalyzed Fenton Haber-Weiss reaction and the generated hydroxyl radicals were rapidly reacted with nitrone spin trap DMPO [9]. The resultant DMPO-OH adducts was detectable with an ESR spectrometer. The compound solution (20 µL) was mixed with DMPO (0.3 M, 20 µL), FeSO4 (10 mM, 20 µL), and H2O2 (10 mM, 20 µL) in a phosphate buffer solution (pH 7.4), and then transferred into a 100 µL quartz capillary tube. After 2.5 min, the ESR spectrum was recorded using an ESR spectrometer. The experimental conditions employed were as follows: magnetic field, 336.5 ± 5 mT; power, 1 mW; modulation frequency, 9.41 GHz; amplitude, 1 × 200; sweep time, 4 min.

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2.8. Superoxide radical scavenging activity Superoxide anion radicals were generated by UV irradiated riboflavin/EDTA system [10]. The reaction mixture containing 0.3 mM riboflavin (20 µL), 1.6 mM EDTA (20 µL), 800 mM DMPO (20 µL) and indicated concentrations of compound solution (20 µL) was irradiated for 1 min under UV lamp at 365 nm. Then, the reaction mixture was transferred to a 100 µL quartz capillary tube of the ESR spectrometer for measurement. The experimental conditions employed were as follows: magnetic field, 336.5 ± 5 mT; power, 10 mW; modulation frequency, 9.41 GHz; amplitude, 1 × 1,000; sweep time, 1 min. 2.9. Peroxyl radical scavenging activity Peroxyl radicals were generated according to the method of Hiramoto et al. [11]. Briefly, 20 µL of 40 mM 2,2’-azobis (2-amidinopropane) dihydrochloride (AAPH) was mixed with 20 µL of phosphate buffered-saline (PBS), 20 µL of 40 mM α-(4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN) and 20 µL of compound solution. The mixture was vortexed and incubated at 37oC for 30 min. Subsequently, reaction mixture was transferred to a sealed capillary tube and spin adduct was recorded with controlled spectrometric conditions; modulation frequency, 100 kHz; microwave power, 10 mW; microwave frequency, 9,441 MHz; magnetic field, 336.5 ± 5 mT and sweep time, 30 sec. 2.10. Culture of cells and viability determination RAW264.7, MRC-5, and HL60 cell were cultured in DMEM and RPMI-1640 containing 5% FBS at 37oC in a 5% CO2 humidified incubator. Cells were grown in 96well plates at a density of 5 × 103 cells/well in the presence of different concentrations of samples. Cell viability was determined by use of the MTT assay [12]. MTT was used as an indicator of cell viability, as determined by mitochondrial-dependent reduction to formazan. In brief, the cells were seeded and then treated with various reagents for the indicated time periods. After various treatments, the medium was removed and the cells were incubated with a solution of 0.5 mg/mL MTT. After incubation for 3 h at 37oC and 5% CO2, the supernatant was removed and the formation of formazan was observed by monitoring the signal at 540 nm, using a microplate reader. 2.11. Determination of intracellular formation of reactive oxygen species (ROS) using DCFH-DA labeling Intracellular formation of reactive oxygen species (ROS) was assessed as described previously using oxidation sensitive dye 2’, 7’-dichlorofluorescin diacetate (DCFHDA) as the substrate [13]. RAW264.7 cells growing in fluorescence microtiter 96-well plates were loaded with

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20 µM DCFH-DA in HBSS and incubated for 20 min in the dark. Nonfluorescent DCFH-DA dye, that is freely penetrated into cells get hydrolyzed by intracellular esterases to 2’, 7’-dichlorofluorescin (DCFH), and tarps inside the cells. Then, cells were treated with different concentrations of test compounds and incubated for 1 h. After washing the cells with PBS three times, 300 µM H2O2 dissolved in HBSS was added to cells. The formation of 2’,7’dichlorofluorescin (DCF), due to oxidation of DCFH in the presence of various ROS, was read after every 30 min at the excitation wavelength (Ex) of 485 nm and the emission wavelength (Em) of 535 nm using a GENions® fluorescence microplate reader (Tecan Austria GmbH, Austria). Dose dependent and time dependent effects of treatment groups were plotted and compared with fluorescence intensity of control and blank groups.

J = 8.78, 2.19 Hz, H-4, 5), 4.21 (4H, t, J = 6.16 Hz, H2-1’ 1’’), 1.66 (4H, m, H-2’, 2’’), 1.25 (4H, m, H-3’, 3’’), 1.25 (4H, m, H-4’, 4’’), 1.25 (4H, m, H2-5’, 5’’), 1.25 (4H, m, H2-6’, 6’’), 1.25 (2H, m, H2-7’), 1.25 (4H, m, H2-8’, 7’’),

2.12. Myeloperoxidase activity Amount of myeloperoxidase (MPO) released by HL60 cell, it was determined by O-dianisidine method with modification. HL60 cells were suspended in RPMI-1640 without phenol red and FBS and seeded into 96-well plates. Cells were preincubated with various concentrations of compounds for 30 min followed by stimulation with TNF-α (0.05 µg/mL) at 37oC for 30 min. Then, cells were added to the assay mixture containing 0.05 mL of 1 mM H2O2 in 0.1 M phosphate buffer (pH 6.0) and 0.05 mL of 0.02 M O-dianisidine (freshly prepared) in deionized water. The amount of MPO released was measured spectrophotometrically at 460 nm and MPO activity was plotted as an absorbance value compared to the untreated blank group. 2.13. Statistical analyses All data are presented as means ± SD. The mean values were calculated based on the data taken from at least three independent experiments conducted on separate days using freshly prepared reagents. Statistical analyses were performed using student’s t-test. Statistical significance was achieved when P < 0.05.

3. Results and Discussion 3.1. Results 3.1.1. Identification structures of compounds (1-3) Compound (1), 2-ethyldecyl 2-ethylundecyl phthalate: 20 yellowish oil; [α] D +0.0030 (c 1.18, CHCl3); IR (neat) γmax 2924, 1721, 2924, 1721, 1604, 1571, 1482, 1462, 1379, 1264, 1113, 1070, 1033, 951, 748, 701/cm; UV (CHCl3) λmax (log ε) 276 (2.95) nm; 1H NMR (400 MHz, CDCl3) δ 7.70 (1H, dd, J = 8.78, 2.19 Hz, H-3, 6), 7.52 (2H, dd,

Table 1. 13C data for compounds 1-3 in CDCl3 Position

1

2

3

1 2 3 4 5 6 7 8 1’ 2’ 3’ 4’ 5’ 6’ 7’ 8’ 9’ 10’ 11’ 12’ 13’ 14’ 15’ 16’ 17’ 18’ 19’ 1’’ 2’’ 3’’ 4’’ 5’’ 6’’ 7’’ 8’’ 9’’ 10’’ 11’’ 12’’ 13’’ 14’’ 15’’ 16’’ 17’’ 18’’ 19’’ 20’’ 21’’

167.8 (s) 132.4 (s) 128.8 (d) 130.9 (d) 130.9 (d) 128.8 (d) 132.4 (s) 167.8 (s) 68.1 (t) 38.7 (d) 30.3 (t) 28.9 (t) 29.7 (t) 29.7 (t) 29.7 (t) 29.3 (t) 31.9 (t) 22.9 (t) 14.2 (q) 23.7 (t) 10.9 (q)

167.7 (s) 132.3 (s) 128.8 (d) 130.9 (d) 130.9 (d) 128.8 (d) 132.3 (s) 167.7 (s) 66.2 (t) 38.7 (d) 31.9 (t) 28.6 (t) 30.3 (t) 29.7 (t) 29.7 (t) 29.7 (t) 27.0 (t) 34.3 (t) 33.1 (d) 36.6 (t) 26.8 (t) 29.2 (t) 22.7 (t) 14.1 (q) 23.7 (t) 11.0 (q) 19.6 (q) 66.2 (t) 38.7 (d) 31.9 (t) 28.9 (t) 30.3 (t) 29.7 (t) 29.7 (t) 29.7 (t) 27.5 (t) 36.5 (t) 39.4 (d) 41.9 (d) 32.5 (t) 32.7 (t) 23.0 (t) 14.2 (q) 23.7 (t) 11.4 (q) 19.1 (q) 26.1 (t) 12.2 (q)

167.8 (s) 132.4 (s) 128.8 (d) 130.9 (d) 130.9 (d) 128.8 (d) 132.4 (s) 167.8 (s) 68.1 (t) 38.7 (d) 30.3 (t) 28.9 (t) 29.7 (t) 22.9 (d) 14.0 (q) 23.7 (t) 10.9 (q)

68.1 (t) 38.7 (d) 30.3 (t) 28.9 (t) 29.7 (t) 29.7 (t) 29.3 (t) 31.8 (t) 22.6 (t) 14.1 (q) 23.7 (t) 10.9 (q)

68.1 (t) 38.7 (d) 30.3 (t) 28.9 (t) 29.7 (t) 22.9 (d) 14.0 (q) 23.7 (t) 10.9 (q)

*Recorded in CDCl3 at 400 MHz (1H) and 100 MHz (13C).


1.25 (4H, m, H2-9’, 8’’), 1.30 (4H, m, H2-10’, 9’’), 0.87 (6H, m, H3-11’, 10’’), 1.39 (4H, m, H2-12’, 11’’), 0.89 (6H, m, H3-13’, 12’’), 13C NMR (100 MHz, CDCl3) δ 167.8 (s, C-1, 8), 132.4 (s, C-2, 7), 128.8 (d, C-3, 6), 130.9 (d, C-4, 5), 6.81 (s, C-1’, 1’’), 38.7 (d, C-2’, 2’’), 30.3 (t, 3’, 3’’), 28.9 (t, C-4’, 4’’), 29.7 (t, C-5’, 5’’, 6’, 6’’, 7’), 29.3 (t, C8’, 7’’), 31.9 (t, C-9’), 31.8 (t, C-8’’), 22.9 (t, C-10’), 22.6 (t, C-9’’), 14.2 (q, C-11’), 14.1 (q, C-10’’), 23.7 (t, C-12’, 11’’), 10.9 (q, C-13’, 12’’) (Table 1), HREIMS m/z 516.4168 [M]+ (calcd for C33H56O4 516.4179, ∆ -1.1 mmu). Compound (2), 2, 12-diethyl-11-methylhexadecyl 2-ethyl20 11-methylhexadecyl-phthalate: yellowish oil; [α] D +0.1o (c 2, MeOH); IR (neat) γmax 2957, 2924, 2855, 1720, 1602, 1575, 1485, 1264, 1115, 1072, 1033, 965, 798, 741/cm; UV (CHCl3) λmax (log ε) 274 (2.39) nm; 1H NMR (400 MHz, CDCl3) δ 7.70 (2H, dd, J = 8.78, 2.18 Hz, H-3, 6), 7.52 (2H, dd, J = 8.78, 2.18 Hz, H-4, 5), 4.27, 4.21 (2H, t, J = 6.25 Hz, H2-1’), 4.27, 4.21 (2H, t, J = 6.25 Hz , H2-1’’), 1.68 (4H, m, H-2’, 2’’, 11’, 11’’), 1.45 (1H, m, H-12’’), 1.33 (2H, m, H2-15’), 1.32 (2H, m, H2-15’’), 1.29 (36H, m, H2-4’, 4’’, 5’, 5’’, 6’, 6’’, 7’, 7’’, 8’, 8’’, 9’, 9’’, 13’, 14’, 14’’, 17’, 17’’, 20’’), 1.25 (12H, m, H2-3’, 3’’, 10’, 10’’, 12’, 13’’), 0.90 (21H, m, H3-16’, 16’’, 18’, 18’’, 19’, 19’’, 21’’); 13CNMR (100 MHz, CDCl3) δ 167.7 (s, C-1, 8), 132.3 (s, C-2, 7), 128.8 (d, C-3, 6), 130.9 (d, C-4, 5), 68.1 (t, C-1’, 1’’), 38.7 (d, C-2’, 2’’), 31.9 (t, C-3’, 3’’), 28.6 (t, C-4’), 30.3 (t, C-5’, 5’’), 29.7 (t, C-6’, 6’’, 7’, 7’’, 8’, 8’’), 27.0 (t, C-9’), 34.3 (t, 10’), 33.1 (d, C-11’), 36.6 (t, C-12’), 26.8 (t, C-13’), 29.2 (t, C-14’), 22.7 (t, C-15’), 14.1 (q, C16’), 23.7 (t, C-17’, 17’’), 11.0 (q, C-18’), 19.6 (q, C-19’), 28.9 (t, C-4’’), 27.5 (t, C-9’’), 36.5 (t, C-10’’), 39.4 (d, C11’’), 41.9 (d, C-12’’), 32.5 (t, C-13’’), 32.7 (t, C-14’’), 23.4 (t, C-15’’), 14.2 (q, C-16’’), 11.4 (q, C-18’’), 19.1 (q, C19’’), 26.1 (t, C-20’’), 12.2 (q, C-21’’) (Table 1); HREIMS m/z 726.6547 [M]+ (calcd for C48H86O4, 726.6526, ∆ + 2.1 nnu). Compound (3), Bis(2-ethylheptyl) phthalate (Fig. 1): 20 Yellowish oil; [α] D 0o (c 0.5, MeOH)); IR (neat) νmax 2924; 1723; 1600, 1577, 1488; 1462; 1379; 1267, 1118, 1071, 1037; 956, 741, 702 cm-1; LREIMS m/z (rel. int.) 418 [M]+ (5), 279 (7), 167 (22), 149 (100), 113 (11), 83 (19), 71 (35), 57 (83); 1H NMR (400 MHz, CDCl3) δ7.70 (2H, dd, J = 8.8, 2.2 Hz, H-3, 6), 7.53 (2H, dd, J = 8.8, 2.2 Hz, H-4, 5), 4.21 (4H, t, J = 6.2 Hz, H2-1, 1), 1.65 (4H, m, H2-2, 2), 1.25 (4H, m, H2-3, 3), 1.29 (8H, m, H2- 4, 4, 5, 5), 1.31 (4H, m, H2-6, 6), 0.89 (6H, m, H3-7, 7), 1.40 (4H, m, H2-8, 8), 0.90 (6H, m, H3-9, 9); 13C NMR (100 MHz, CDCl3) data, see Table 1. 3.1.2. Effect of compounds (1-3) on lipid peroxidation inhibition The lipid peroxidation inhibition activities of compounds

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Compound (1). 2-ethyldecyl 2-ethylundecyl phthalate

Compound (2). 2, 12-diethyl-11-methylhexadecyl 2-ethyl-11methylhexadecylphthalate

Compound (3). Bis (2-ethylheptyl) phthalate

Fig. 1. Structures of compounds (1-3).

(1-3) were determined by the FTC method. The FTC method was used to measure the peroxide level during the initial stage of lipid oxidation. The effects of compounds (1-3), in preventing the peroxidation of linoleic acid, are shown in Fig. 2. After the incubation period (7 day), the formation of peroxides was stopped because of nonavailability of linoleic acid. Also, the intermediate products had been converted to stable end-products resulting in the stoppage of oxidation of Fe2+. Therefore, lower absorbance indicates a higher level of antioxidant activity. The compound 2 (79.5%) and α-tocopherol (83.5%) exhibited higher activity than that of compound 1 (67.3%) and compound 3 (44.7%) compared to the control after 7 days. 3.1.3. Free radical scavenging activity of compounds (1-3) Compound (1-3) exhibited a potent free radical scavenging activities against DPPH, hydroxyl, superoxide and peroxyl radicals observed by electron spin resonance (ESR) spectrometer, with IC50 values of 1.38 mM (1), 0.82 mM (2) and 4.38 mM (3) for DPPH, 0.6 mM (1), 0.38 mM (2) and 3.25 mM (3) for hydroxyl, 0.7 mM (1), 0.49 mM (2) and 8.76 mM (3) for superoxide, and 0.99 mM (1),

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Fig. 2. Antioxidative activities of compounds in a linoleic acid emulsion system, measured by the ferric thiocyanate method. The control is defined where no antioxidant is added in the antioxidative activity test. Table 2. IC50 values of compounds against free radicals Radical scavenging activity (IC50, mM) Radical DPPH Hydroxyl Superoxide Alkyl

Compounds 1

2

3

1.38 0.60 0.70 0.99

0.82 0.38 0.49 0.58

4.38 3.25 8.76 8.90

Radical scavenging activity (%) = [(Blank peak area - Sample peak area)/Blank peak area] × 100 (%).

0.58 mM (2) and 8.9 mM (3) for peroxyl radical scavenging activity, respectively (Table 2). 3.1.4. Effects of compounds on intracellular reactive oxygen species (ROS) level generated by hydrogen peroxide in RAW 264.7 and cytotoxic effects Cytotoxic effects of compounds (1-3) were evaluated on mouse macrophage (RAW 264.7) and human lung fibroblast (MRC-5), and the results showed that compounds (1-3) did not exhibit cytotoxic effects at the tested concentrations (Figs. 3A and 3B). The compounds (1-3) effectively quenched with free radicals, generated various methods in this study. Therefore, we were interested in studying the direct effects of these compounds to scavenge cellular radicals. As shown in Figs. 4A, 4B, and 4C, fluorescence emitted by DCF following ROSmediated oxidation of DCFH followed a time course increment up to 200 min. Pre-treatment with the compounds (1-3) decreased the DCF fluorescence dose- and time-

Fig. 3. Cytocompatible effects of compounds on RAW 264.7 cells (A) and MRC-5 cells (B). Different concentrations of compounds were applied to the cells for 24 h and cell viability was assessed by MTT assay, as described in the text. Results are means ± standard error of three independent experiments.

dependently. The compounds (1-3) exerted a considerable radical scavenging effect at 100 µg/mL concentration after 30 min. More clearly, compound (3) could scavenge radicals significantly throughout the incubation time than another compound (2, 3). 3.1.5. Myeloperoxidase (MPO) inhibitory effects of compound (1-3) Myeloperoxidase the most abundant haem enzyme in neutrophils, converts H2O2 into HOCl which is the most powerful oxidant acting against pathogens during infections. The large amount of ROS released into the extracellular medium by neutrophils not only contributes to kill


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Fig. 5. Inhibition of myeloperoxidase (MPO) activity in HL60 cells by compounds. Cells were treated with different concentrations of compounds and expression of MPO was stimulated by human TNF-α (0.05 µg/mL). An assay mixture containing 2 mM H2O2 and 0.02 M O-dianisidine was added to the cells and amount of MPO released was measured spectrophotometrically at 460 nm. MPO activity was compared with TNF-α non-stimulated blank and sample non-treated control group. Results are means ± standard error of three independent experiments.

Fig. 4. Cellular radical scavenging activity of compounds. RAW264.7 cells were labeled with non-toxic fluorescence dye, DCFH-DA, and treated with different concentrations of compounds. Fluorescence intensities of DCF due to oxidation of DCFH by cellular ROS (generated by H2O2) were detected time-dependently (Ex = 485 nm and Em = 535 nm). Effects of compounds on the scavenging of cellular ROS were compared with H2O2 nonstimulated blank and sample non-treated control groups in three independent experiments: (a). Compound (1). 2-ethyldecyl 2ethylundecyl phthalate; (b). Compound (2). 2, 12-diethyl-11methylhexadecyl 2-ethyl-11-methylhexadecylphthalate; (c). Compound (3). Bis (2-ethylheptyl) phthalate.

pathogens, but also damages healthy tissues of the host. This causes oxidation of phthalate compounds in proteins and unsaturated fatty acids of membrane lipids destabilizing the cell membrane integrity. Therefore, inhibition of MPO activity is an important approach to control free radicalmediated oxidation of biomolecules in neutrophils. In this study, we evaluated whether compounds can inhibit MPO activity which in turn helps to prevent oxidation of cellular biomolecules. To this purpose, human myeloid cell line HL60 was selected due to its reported high expression of MPO following stimulation. As depicted in Fig. 5, compounds could reduce MPO activity dose-dependently compared to TNF-α-stimulated control in HL60 cells. However, compound 3 could not inhibit MPO as efficiently as compound 1, 2. At the 200 µg/mL concentration of compound 1, 2, and 3, 31.02, 43.5, and 6.07% inhibition of MPO were observed and its can be speculated that this is an indirect way of acing as cellular antioxidant. 3.2. Discussion S. Hippocampus Kuda, is a well-known TCM with a long history of use to treat various diseases including anti-aging, anti-tumor, anti-fatigue, hormone-like activity, boosting hematopoiesis function and Ca2+ channel blocking activities. However, the reports related to natural products from

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seahorse are published rarely. Our present study resulted in the isolation of two new phthalate derivatives (1 and 2) and one known derivative (3) from the methanol extracts of Hippocampus Kuda, and the structural elucidation of three compounds were carried out mainly by the comprehensive interpretation of 1D and 2D NMR data. Most of phthalate esters derivatives were chemically synthesized for the plasticizer usage due to its flexibility property previously [16]. The latest studies of phthalate derivatives revealed a number of variously novel biological activities and mechanism besides toxicities such as antileukemic, anti-mutagenic, antibacterial, antiviral, antiandrogen, and hormone-like activities [14-18]. Moreover, phthalate derivative has been isolated from natural resource like marine bacterial [19,20], plants [15], marine algae [16,21,22], marine animal, and others. The further research provided the credible evidence that phthalate derivatives can be occurred naturally because of 13C isotope content, instead of 14C in artificial products. Therefore, these results have increasingly attracted special attention from scientists. In order to assess the biological activities of these compounds, inhibition activity of free radical-mediated oxidation was employed by lipid peroxidation inhibition assay, free radical scavenging activity, and intracellular reactive oxygen species (ROS) level, respectively. In in vitro systems, various ROS are generated and can cause cell damage. A major form of cellular oxidative damage is lipid peroxidation, which is initiated by ROS through the extraction of a hydrogen atom from unsaturated fatty acids of membrane phospholipids [23]. Membrane lipids are particularly susceptible to oxidation, not only because of their high polyunsaturated fatty acid content, but also because of their association in the cell membrane with enzyme and non-enzyme systems capable of generating free radical species [24]. Peroxidation of membrane lipid is a cardinal feature of oxy-radical toxicity [25]. The chain reaction of lipid peroxidation yields several types of secondary free radicals and a large number of reactive compounds, resulting in the destruction of cellular membranes and other cytotoxic responses [26]. Oxidative degradation of polyunsaturated fatty acids occurs in two sequential steps [27]. The initiation reaction involves reactive oxygen species such as hydroxyl radical as initiators, forming a conjugate-stabilized, carbon centered radical (L.). This reacts rapidly with oxygen to form peroxy radical (LOO.), which abstracts a hydrogen atom from another fatty acid to form lipid hydroperoxides (LOOH) and a new carbon centered radical (L), until the chain reaction is terminated (propagation). Therefore, antioxidative materials acting in living systems are classified as preventive antioxidants and chain-breaking [28]. Some of compounds prevented the formation of lipid peroxide and scavenged superoxide anions. They would be

classified as preventive antioxidant. Others strongly inhibited lipid peroxidation but showed little effect on superoxide. They would be classified as chain-breaking ones. First, to investigate a direct scavenging effect of these compounds, peroxidation inhibitory activity, DPPH radical, hydroxyl radical, superoxide radical and peroxyl radical were generated in emulsion system and analyzed by electron spin trapping technique (ESR). The scavenging activity from compound 2 revealed the highest activity, then that of the other compounds. Based on above results, radical scavenging effects of compounds were examined in a live cell system, following treatment of RAW 264.7 cell line with hydrogen peroxide. Intracellular ROS was measured using DCFHDA that has widely used to monitor intracellular oxidant stress. The results obtained from this cell system were consistent with that of a cell-free system. We could also observe a dose-dependent effect of compounds on antioxidant effect in live cell system. According to 1H and 13C (Table 1), compound 1 revealed the presence of four methyl groups, nineteen methylene units (two O-bearing methylene), four aromatic carbons, two methene, two quaternary carbons, and two carbonyl carbons. The IR spectrum showed absorptions for alkane (2924/cm, C-H stretch), ester (1721/cm, C=O), aromatic (1604, 1571, 1482/cm, C=C), ester (1264/cm, C-O) functionalities. Compound 2 (Data not shown) revealed the presence of seven methyl groups, twenty-eight methylene units (two O-bearing methylene), four aromatic carbons, five methines, two quaternary carbons, and two carbonyl carbons. Compound 3 contained two carbonyl carbons, four aromatic methines, two O-brearing methelene, four methyl, twelve alkyl methylene, and two alkyl methane groups. To elucidate the scavenging radical mechanisms for these compounds described above, we investigated the relative phthalates derivatives reported previously. The results revealed that the phenyl ring hydroxylation of phthalate derivatives would be possible under normal environmental conditions into the ring 4-hydroxylated derivatives, the hydroxylated derivatives of phthalates (DEP and DBP) also exhibited estrogenic activity with an estrogen receptors (ER) binding assay [29]. These estrogenic activities of phthalate-4OH are supported by a structure-activity relationship study, which shows that ER ligands usually contain a phenolic ring in their structures [30]. The phenolic rings in these structures have the antioxidative activity and free radical scavenging activity based on mayrecent reports. In our present study, the difference on the antioxidant and free radical scavenging activities among these three compounds was interpreted as following, compound 2 showed the relatively high activity compared with the others, the most reasonable explanation would like to go to the instable side long-chains [seven methyl groups, twenty eight methylene


units (two O-bearing methylene), four aromatic carbons, five methines, and two carbonyl carbons], which make the hydroxylation more easily. It is the first report for the structure-activity relationship of these two new phthalate derivatives with antioxidant activity and free radical scavenging activity, as well as the known compound (3 compounds).

4. Conclusion In conclusion, the methanol extracts of fresh Hippocampus Kuda led to the new isolations 2-ethyldecyl 2-ethylundecyl phthalate (1) and 2, 12-diethyl-11-methylhexadecyl 2-ethyl11-methylhexadecylphthalate (2), as well as a natural known Bis (2-ethylheptyl) phthalate (3) firstly obtained, respectively. They were tested for their antioxidant activities, and the compounds (2) exhibited higher antioxidant activity than other compounds (1) and (3). Thus these compounds have noteworthy potential for application as antioxidants in functional food, cosmetics, and pharmaceutical industries. Meanwhile, additional studies on the mechanisms and in vivo are highly warranted to achieve a better understanding of important antioxidant properties.

Acknowledgment This research was supported by a grant from Marine Bioprocess Research Center of the Marine Bio 21 Center funded by the Ministry of Maritime Affairs and Fisheries, Republic of Korea.

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