|Year : 2012 | Volume
| Issue : 30 | Page : 98-102
Chemical constituents with free-radical-scavenging activities from the stem of Fissistigma polyanthum
Hua Fan1, Tong Zheng1, Yu Chen2, Guang-Zhong Yang1
1 Department of Natural Products, College of Pharmacy, South Central University for Nationalities, Wuhan, P.R., China
2 College of Chemistry and Material Sciences, South Central University for Nationalities, Wuhan, P.R, China
|Date of Submission||19-Apr-2011|
|Date of Acceptance||15-Jun-2011|
|Date of Web Publication||23-May-2012|
College of Pharmacy, South Central University for Nationalities, Wuhan 430074, P. R.
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background: Fissistigma polyanthum is a liane belonging to the Annonaceae family and it is one of the most important crude drugs in traditional Chinese medicine. Objective: The objective was to describe the structural elucidation and the free-radical-scavenging activities of the isolated compounds from Fissistigma polyanthum. Material and Methods: The chemical constituents were isolated and purified by normal, reverse column chromatography and HPLC. Their structures were identified by spectroscopic methods ( 1 H NMR and 13 C NMR) and by comparison with literature values, and the free-radical-scavenging activities of these two compounds were also evaluated through three in vitro model systems (DPPH, trolox equivalent antioxidant capacity (TEAC) and Co (II) EDTA-induced luminol chemiluminescence by flow injection). Results: Two known compounds, named kanakugiol (1) and teutenone A (2), were isolated from the stem of Fissistigma polyanthum for the first time, and compound 1 exhibited moderate free-radical-scavenging activity. Conclusion: Fissistigma polyanthum, which has traditionally been used as an important Chinese medicine, showed a certain free-radical-scavenging activity.
Keywords: Co (II) EDTA-induced luminol chemiluminescence by flow injection, DPPH, Fissistigma polyanthum, free-radical-scavenging activities, TEAC
|How to cite this article:|
Fan H, Zheng T, Chen Y, Yang GZ. Chemical constituents with free-radical-scavenging activities from the stem of Fissistigma polyanthum. Phcog Mag 2012;8:98-102
|How to cite this URL:|
Fan H, Zheng T, Chen Y, Yang GZ. Chemical constituents with free-radical-scavenging activities from the stem of Fissistigma polyanthum. Phcog Mag [serial online] 2012 [cited 2020 Nov 24];8:98-102. Available from: http://www.phcog.com/text.asp?2012/8/30/98/96549
| Introduction|| |
The genus Fissistigma, belonging to the family Annonaceae, comprises almost 75 species worldwide, spreading across Africa, Oceania, and Asia. In China, there are 23 species. Previous phytochemical investigations revealed in the genus the presence of alkaloids, ,,, cyclopentenones,  furanone,  and flavones,  which exerted diverse bioactivities such as liver protection, anti-inflammatory, anti-arthritic, and anti-tumor effects.  Fissistigma polyanthum is a liane that is abundant in China, India, Burma, and Vietnam. It has been traditionally used to prepare herbal medicines, while limited numbers of reports concerning the chemical constituents and biological activities of Fissistigma polyanthum have appeared in the literature.
Reactive oxygen species, such as peroxyl radical, superoxide anion radical, and hydroxyl radical, are constantly generated in vivo both by aerobic metabolism and exogenous sources such as UV radiation, environmental pollution, and the diet. The formation of reactive oxygen species may cause oxidative stress and destruction of unsaturated lipids, DNA, proteins, and other essential molecules, which leads to aging and the pathogenesis of such degenerative or chronic diseases as arteriosclerosis and cancer. , Therefore, the antioxidants which can prevent oxidative reactions in biological tissues against molecular targets are essential for the normal metabolism of human body. However the use of synthetic antioxidants, such as butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT), which are widely used nowadays in processed food products, is now in doubt due to safety concerns about their potential toxicity and unwanted side effects. , Thus, more and more attention is increasingly being focused on the development and utilization of natural sources as antioxidants. , To date, there have been few reports addressing the evaluation of the free-radical-scavenging activities of the stem of F. polyanthum. Thus, it is necessary to conduct further research on the chemical constituents with free-radical-scavenging activities from this plant.
| Materials and Methods|| |
Fissistigma polyanthum were collected from Xishuangbanna prefecture, Yunnan province, P. R. China and identified by Xishuangbanna prefecture National medicine Research Institute. The voucher specimen was deposited in the Herbarium of the College of Pharmacy, South Central University for Nationalities.
The 1 H- and 13 C-NMR spectra were measured on a Bruker-AM-400 NMR spectrometer at room temperature, using TMS as an internal standard. Chemical shifts (δ) were expressed in parts per million (ppm), with the coupling constants (J) reported in Hertz (Hz). Column chromatographies were carried out with silica gel 60 H (200-300 mesh, Qingdao Haiyang Chemical Group Co., China), C18 reversed-phase silica gel (YMC Co., Ltd., Japan) and Sephadex LH-20 (Pharmacia), TLC was performed on a precoated silica gel GF254 plate (Qingdao Haiyang Chemical Group Co., China), with spots detected by UV254 and anisaldehyde/H 2 SO 4 (10%). 1, 1-diphenyl-2- picrylpicrylhydrazyl(DPPH), 2, 2'-azino-bis(3-ethylbenzoth- iazoline-6-sulfonate) (ABTS) and 6-hydroxy-2, 5, 7, 8-tetramethylchroman-2-carboxylic acid (Trolox) were obtained from Sigma-Aldrich. Gallic acid and ascorbic acid (V C ) were purchased from the Chemical Company, Shanghai, P. R. China. Other chemicals and solvents in this experiment were all analytical grade or higher and from Shanghai Chemical Reagent Co.
The dried and powered stems of Fissistigma polyanthum (876 g) were extracted with 95% methanol (MeOH) three times at room temperature. The MeOH extract was evaporated under vacuum to dryness as a dark brown mass (103 g) and then the concentrated MeOH extract was suspended in 90% H 2 O/methanol (MeOH). The solution was successively partitioned with petroleum ether (P.E.), ethyl acetate (EtOAc), and n-BuOH.
The combined P.E. and EtOAc extracts (26 g) were chromatographed on a silica gel with P.E.- acetone (Me 2 CO) (95:5, 9:1, 8:2, 7:3, 6:4, 0:1, v/v) to provide eleven fractions (Fr.1-Fr.11). Fr.7 (2.067 g) was subjected to column chromatography (silica gel, cyclohexane/EtOAc 95:5, 9:1, 8:2, v/v) to give eleven fractions (Fr.7.1-Fr.7.11). Fr.7.6 (336 mg) was subjected to column chromatography (ODS, H 2 O/MeOH 7:3, 6:4, 1:1, 3:7) to give compound 1 (10 mg). Fr.7.10 (226.2 mg) was subjected to column chromatography (ODS, H 2 O/MeOH 7:3, 6:4, 1:1, 4:6, 3:7, 2:8) to give compound 2 (14.3 mg).
Spectral data of the purified compounds isolated from Fissistigma polyanthum [Figure 1].
|Figure 1: Structures of the compounds (1 and 2) isolated from Fissistigma polyanthum|
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Compound 1 , yellow oil, 1 H-NMR (400 MHz, CD 3 OD): δ8.22 (1H, d, J = 16 Hz, H-α), 8.14 (1H, d, J = 16 Hz, H-β), 8.01 (1H, d, J = 7.6 Hz, H-2), 7.54 (1H, t, J = 7.6 Hz, H-3), 7.63 (1H, m, H-4), 7.54 (1H, t, J = 7.6 Hz, H-5), 8.01(1H, d, J = 7.6 Hz, H-6), 3.84 (6H, s, 2×OCH3), 4.18 (3H, s, -OCH3), 3.90 (3H, s, -OCH3); 13 C-NMR (100 MHz, CD 3 OD) δ138.8 (C-1),128.0 (C-2),128.3 (C-3),132.4 (C-4), 128.3 (C-5), 128.0 (C-6), 111.6 (C-1'), 149.0 (C-2'), 137.3 (C-3'), 149.5 (C-4'), 137.1(C-5'), 150.2 (C-6'), 123.0 (C-α), 138.7 (C-β), 192.8 (C=O), 60.3 (2×OCH 3 ), 60.4 (-OCH 3 ), 60.6 (-OCH 3 ), 1 H and 13 C-NMR data were identical to those recorded in reference. 
Compound 2 , colorless needle, 1 H-NMR (400 MHz, CD 3 OD): δ1.31(3H, s), 1.42 (3H, s), 5.95 (1H, s, H-6); 13 C-NMR (100 MHz, CD 3 OD): δ 39.8 (C-1), 17.0 (C-2), 40.0 (C-3), 70.2 (C-4), 171.6 (C-5), 122.4 (C-6), 202.3 (C-7), 41.0 (C-8), 33.5 (C-9), 35.7 (C-10), 23.3 (C-14), 27.5 (C-15), 1 H and 13 C-NMR data were identical to those recorded in reference. 
The free-radical-scavenging activities of the purified compounds were evaluated through 1, 1-diphenyl-2-picrylhydrazyl (DPPH) method
Scavenging activities of the purified compounds from F. polyanthum toward DPPH radical were assessed by using the method described by Scherer and Godoy with a slight modification. , Briefly, a 0.08 mM solution of DPPH radical solution in methanol was prepared and then the purified compounds at different concentrations (0.1 ml) were added to the prepared DPPH radical solution (3.9 ml); the mixture was shaken vigorously, after a 30-minute incubation period at 37°C in the dark, the absorbance was measured at 517 nm by using a UV-visible spectrophotometer. Obviously, decreasing of the DPPH solution absorbance indicates an increase of the DPPH radical-scavenging activity. The radical scavenging activity was given as DPPH radical scavenging effect that is calculated in equation (1):
DPPH radical scavenging effect (%) = [(A 0 -A 1 )/A 0 ] × 100
where A 0 was the absorbance of control and A 1 was the absorbance in the presence of the standard or purified compounds at different concentrations. Ascorbic acid (V C ) and gallic acid severed as positive controls, respectively. All the tests were performed in triplicate. The scavenging activities of the purified compounds toward DPPH radical were expressed as IC 50 , which was determined to be effective concentration at which DPPH radical was scavenged by 50%. The IC 50 value was obtained by interpolation from linear regression analysis.
The free-radical-scavenging activities of the purified compounds were evaluated through TEAC method
Scavenging activities of the purified compounds from F. polyanthum toward ABTS radical were also measured. ,, Briefly, a stock solution of ABTS radical cation was prepared by dissolving ABTS (7 mM, 25 ml in deionised water) with potassium persulfate (K 2 S 2 O 8 ) (140 mM, 440 μl). The mixture was left to stand in the dark at room temperature for15-16 hours (the time required for formation of the radical) before use. For the evaluation of ABTS radical scavenging activity, the working solution was prepared by the previous solution and diluting it in ethanol to obtain the absorbency of 0.700 ± 0.02 at 734 nm (ABTS working solution should be replaced every 5 days at least because the free radical is easy to degrade). The purified compounds (0.1 ml) at different concentrations were mixed with the ABTS working solution (1.9 ml) and the reaction mixture was allowed to stand at 30°C for 6 minutes; then the absorbance was measured by using a UV-visible spectrophotometer at 734 nm, at which point the antioxidants present in the purified compounds began to inhibit the radical, producing a reduction in absorbance, with a quantitative relationship between the reduction and the concentration of antioxidants present in the tested sample. The radical scavenging activity was given as ABTS radical scavenging effect that is calculated in the equation (2):
ABTS radical scavenging effect (%) = [(A 0 -A 1 )/A 0 ] × 100
At the same time a standard curve was obtained using trolox standard solution at various concentrations (ranging from 0 to 100 μg/ml) in 95% ethanol. Scavenging activities of the purified compounds toward ABTS radical were expressed as TEAC (trolox equivalent antioxidant capacity). Different concentrations of each purified compound were chosen to test the ABTS radical scavenging activity. The results were compared with the standard curve for calculation of TEAC.
Ascorbic acid (V C ) and gallic acid were used for positive controls, respectively. All the tests were performed in triplicate.
The free-radical-scavenging activities of the purified compounds were evaluated through Co (II) EDTA-induced luminol chemiluminescence by flow injection method
Scavenging activities of the purified compounds from F. polyanthum toward hydroxyl radical were assessed by Co (II) EDTA-induced luminol chemiluminescence by the flow injection method described by Giokas, Vlessidis, and Evmiridis with some modifications. ,, Briefly, the FIA (flow injection analysis) manifold was designed. Once switching on the pump, the Co (II)-stream (7.12 × 10 -4 mol/l, pH = 9.0 ± 0.03), luminol-stream (2.28 × 10 -4 mol/l, pH = 9.0 ± 0.03) and carrier-stream were initially mixed in order to reach a stable background. A stable baseline was obtained by mixing the H 2 O 2 -regent stream (0.8 × 10 -3 mol/l, pH = 9.0 ± 0.03) into the system when Cobalt (II) ion increased the chemiluminescene signal of luminol-H 2 O 2 system. A loss of signal (negative peak) was observed which corresponded to the hydroxyl radical scavenging activity of the antioxidant when the antioxidant was injected to the system. Thus, the effect of antioxidant was measured by the depression of the signal from its initial level (uninhibited) and expressed as hydroxyl radical scavenging effect, calculated as follows:
Hydroxyl radical scavenging effect (%) = [height of negative peak/ (baseline-background)] × 100
Each purified compound was injected three times on time intervals of 40 seconds at least. Ascorbic acid (V C ) and gallic acid were used for positive controls, respectively. The scavenging activities of the purified compounds toward hydroxyl radical were also expressed as IC 50 .
Statistical analyses of results of activity studies
The results were performed as mean ± standard deviation (SD) of three determinations. Analysis of significance differences among means were tested by one-way analysis of variance. The IC 50 values were calculated by linear regression analysis.
| Results and Discussion|| |
The1, 1-diphenyl-2-picrylhydrazyl (DPPH), 2, 2'-azino-bis(3-ethylbenzothiazoline-6-sulonate) (ABTS) and hydroxyl radical-scavenging activities of the purified compounds of F. polyanthum are shown in [Table 1].
|Table 1: In vitro antioxidant activities of the purified compounds (1-2) from Fissistigma polyanthum|
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The IC 50 values of DPPH radical scavenging activity decreased as follows: gallic acid > V C > 1 > 2 . The DPPH radical is considered to be a model of lipophilic radical in this study. In the presence of an antioxidant, this can donate an electron to DPPH radical, the purple color typical of free DPPH radical decay. Thereafter the DPPH radical receives a proton from the antioxidant and become a protonated DPPH species, which can be followed spectrophotometrically in absorbance at 517 nm.
The trolox equivalent antioxidant capacity (TEAC) assay is widely used applied to assess the amount of radicals that can be scavenged by an antioxidant, which is based on the antioxidant ability (in terms of radical-scavenging capacity) to react with ABTS + generated in the system. The TEAC value is assigned by comparing the scavenging capacity of an antioxidant to that of trolox and a high TEAC value indicated a high level of antioxidant activity.  From the TEAC values listed in [Table 1], the ABTS radical scavenging activity of the tested compounds was in a decreasing order: gallic acid > V C > 1 > 2 , which was consistent with the results of the DPPH assay.
Scavenging activities of the purified compounds toward hydroxyl radical was measured by Co (II) EDTA-induced luminol chemiluminescence by flow injection method which based on the catalytic oxidation of hydrogen peroxide by luminol-H 2 O 2 -Co (II)-EDTA system, forming a hydroxyl radical flux that can produce a stable chemiluminescence signal which was attenuated in the presence of antioxidants. As shown in [Table 1], the hydroxyl radical scavenging activity of the tested compounds decreased in the order: gallic acid > V C > 1 > 2 , which correlated highly with the results measured by the DPPH and TEAC assays.
It was observed that radical-scavenging activity of the purified compounds of F. polyanthum decreased with the order: gallic acid > V C > 1 > 2 in all three tests mentioned above, although the potency of the compounds was quite different in the three assays. Among the tested compounds, compound 1 with a phenolic hydroxyl group exerted moderate antioxidant activity, while compound 2 with no phenolic hydroxyl group showed a very weak capacity. These results supported the idea that free-radical-scavenging activity can be attributed to the number of protons available for donation by free hydroxyl groups, and the phenolic hydroxyl structural group in benzene ring contributes much to the free-radical-scavenging activity. 
| Conclusion|| |
Chromatographic separation of the P.E. and EtOAc extracts of F. polyanthum, using a normal-phase and reverse-phase silica gel column chromatography, yielded two natural products: kanakugiol ( 1 ) and Teutenone A ( 2 ), which were isolated from F. polyanthum for the first time. The compounds were identified by spectroscopic methods ( 1 H NMR and 13 C NMR) and by comparison with literature values. The free-radical-scavenging activities of these two compounds were also determined by comparing their free-radical-scavenging effects through not only DPPH and TEAC, but also through the more sensitive and convenient Co ( II ) EDTA-induced luminol cheniluminescence by flow injection test. Among them, compound 1 with a phenolic hydroxyl group exhibited moderate antioxidant activity (TEAC = 0.38 ± 0.01) against DPPH and hydroxyl radicals with IC 50 values of 90.69 μM and 3.85 μM, respectively and therefore may be a promising natural antioxidant. Further research on isolation and identification of more bioactive compounds from F. polyanthum will be helpful to understand this traditional herbal medicine.
| References|| |
|1.||Wu JB, Cheng YD, Kuo SC, Wu TS, Iitaka Y, Ebizuka Y, et al. Fissoldhimine, a novel skeleton alkaloid from Fissistigma oldhamii. Chem Pharm Bull 1994;42:2202-4. |
|2.||Wu JB, Cheng YD, Chiu NY, Huang SC, Kuo SC. A Novel Morphinandienone Alkaloid from Fissistigma oldhamii. Planta Med 1993;59:179-80. |
|3.||Chia YC, Chang FR, Teng CM, Wu YC. Aristolactams and dioxoaporphines from Fissistigma balansae and Fissistigma oldhamii. J Nat Prod 2000;63:1160-3. |
|4.||Deng Y, Chen J, Wu FE. Two new aporphine alkaloids fom Fissistigma bracteolatum. Chin Chem Lett 2002;13:862-4. |
|5.||Chia YC, Wu JB, Wu YC. Two novel cyclopentenones from Fissistigma odhamii. Tetrahedron Lett 2000;41:2199-201. |
|6.||Chia YC, Chang FR, Wu YC. Fissohamione, A novel furanone from Fissistigma odhamii. Tetrahedron Lett 1999;40:7513-4. |
|7.||Alias Y, Awang K, Hadi AH, Thoison O, Sévenet T, Païs M. An antimitotic and cytotoxic chalcone from Fissistigma lanuginasum. J Nat Prod 1995;58:1160-6. |
|8.||Duthie GG, Duthie SJ, Kyle JA. Plant polyphenols in cancer and heart disease: implications as nutritional antioxidants. Nutr Res Rev 2000;13:79-106. |
|9.||Lobo V, Patil A, Phatak A, Chandra N. Free radicals, antioxidants and functional foods: Impact on human health. Phcog Rev 2010;4:118-26. |
|10.||Ito N, Fukushima S, Hagiwara A, Shibata M, Ogiso T. Carcinogenicity of butylated hydroxyanisole in F344 rats. J Natl Cancer Inst 1983;70:343-52. |
|11.||Wichi HP. Enhanced tumor development by butylated hydroxyanisole (BHA) from the perspective of effect on forestomach and oesophageal squamous epithelium. Food Chem Toxicol 1988;6:717-23. |
|12.||Ozen T, Turkekul I. Antioxidant activities of Sarcodon imbricatum wildly grown in the Black Sea Region of Turkey. Pharmacogn Mag 2010;6:89-97. |
|13.||Patel V, Shukla S, Patel S. Free Radical Scavenging Activity of Grangea maderaspatana Poir. Pharmacogn Mag 2009;5:381-7. |
|14.||Lee SM, Baek SH, Lee CH, Lee HB, Yee YH. Cytotoxicity of lignans from Lindera erytherocarpa Makino. Nat Prod Sci 2002;8:100-2. |
|15.||Fraga BM, Hernandez MG, Mestres T, Terrero D, Arteaga JM. Nor-sesquiterpenes from Teucrium heterophyllum. Phytochemistry 1995;39:617-9. |
|16.||Scherer R, Godoy HT. Antioxidant activity index (AAI) by the 2, 2-diphenyl-1-picrylhydrazyl method. Food Chem 2009;112:654-8. |
|17.||Alma MH, Mavi A, Yilderim A, Digrak M, Hirata T. Screening chemical composition and in vitro antioxidant and antimicrobial activities of the essential oils from Origanum syriacum L. growing in Turkey. Biol Pharm Bull 2003;26:1725-9. |
|18.||Zulueta A, Esteve MJ, Frigola A. ORAC and TEAC assays comparison to measure the antioxidant capacity of food products. Food Chem 2009;114:310-6. |
|19.||Re R, Pellegrini N, Proteggente A, Pannala A, Yang M, Rice-Evans C. Antioxidant activity applying an improved ABTS radical cation decolourisation assay. Free Radic Biol Med 1999;26:1231-7. |
|20.||Arts MJ, Dallinga JS, Voss HP, Haenen GR, Bast A. A critical appraisal of the use of the antioxidant capacity (TEAC) assay in defining optimal antioxidant structures. Food Chem 2003;80:409-14. |
|21.||Giokas DL, Vlessdis AG, Evmiridis NP. On-line selective detection of antioxidants free-radical scavenging activity based on Co (II) EDTA-induced luminol chemiluminescence by flow injection analysis. Anal Chim Acta 2007;589:59-65. |
|22.||Sariahmetoglu M, Wheatley RA, Cakýcý Y, Kanzýk Y, Townshend A. Evaluation of the antioxidant effect melatonin by flow injection analysis-luminol chemiluminescence. Pharmacol Res 2003;48:361-7. |
|23.||Sariahmetoglu M, Wheatley RA, Cakici I, Kanzik I, Townshend A. Flow injection analysis for monitoring antioxidants effects on luminol chemiluminescence of reactive oxygen species. Anal Lett 2003;36:749-65. |
|24.||Meyer AS, Frankel EN. Antioxidant activity of hydroxycinnamic acids on human low-density lipoprotein oxidation. Methods Enzymol 2001;335:256-65. |
|25.||Shaidi F, Anitha PK, Wanasundara PD. Phenolic antioxidants. Crit Rev Food Sci Nutr 1992;2:67-103. |