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Original Article Antioxidants in Aerial Parts of Hypericum Sampsonii, Hypericum Japonicum and Hypericum Perforatum

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Original Article Antioxidants in Aerial Parts of Hypericum Sampsonii, Hypericum Japonicum and Hypericum Perforatum International Journal of Food Science and Technology 2009, 44, 2249–2255 2249 Original article Antioxidants in aerial parts of Hypericum sampsonii, Hypericum japonicum and Hypericum perforatum Chung Li Chen, Chien Hsiu Huang & Jih Min Sung* Department of Agronomy, National Chung Hsing University, Taichung, Taiwan, 40227, ROC & Department of Biotechnology, Hung Kuang University, Taichung County, Taiwan, 43302 (Received 19 March 2009; Accepted in revised form 14 August 2009) Summary Antioxidants contents and antioxidative enzymes and their activities in fresh aerial tissues of Hypericum sampsonii (Sampson’s St John’s Wort), Hypericum japonicum (Japanese St John’s Wort) and Hypericum perforatum were investigated. Hypericum sampsonii contained more total ascorbate [34.33 lmol g)1 fresh weight (FW)] than H. perforatum (57% less) and H. japonicum (82% less). It also contained more thiol and phenolics than two other species. Hypericum japonicum had highest superoxide dismutase (SOD) activity (8.74 mmol min)1 g)1 FW), followed by H. sampsonii (2% less) and H. perforatum (37% less). Hot-air dried H. perforatum materials contained more thiol [208.7 lmol g)1 dry weight (DW)] and phenolics (352.82 mg g)1 DW) than freeze-dried and fresh materials. Both drying treatments decreased the activities of antioxidative enzymes in aerial tissues of H. perforatum. However, freeze-dried H. perforatum contained the highest SOD activity (5.42 mmol min)1 g)1 DW) among the antioxidative enzymes measured from both freeze-dried and hot-air dried tissues (ranged from 0.02 to 2.65 lmol min)1 g)1 DW). Keywords Antioxidant, drying, Hypericum japonicum, Hypericum perforatum, Hypericum sampsonii. antioxidative compounds may be of importance in Introduction preventing or reducing the ROS-related damages, pro- Reactive oxygen species (ROS) are inevitably produced vided that they have protective mechanism against the by many redox processes that occurred in human digestive process (Muth et al., 2004; Vouldoukis et al., organisms, and they serve important physiological 2004). functions (Urso & Clarkson, 2003; Vasdev et al., The genus Hypericum, which contains more than 400 2006). However, ROS also have deleterious effects on species, is widespread in Europe, North America, North human health since they may initiate or develop a wide Africa and West Asia. Many species of genus Hypericum range of diseases (e.g. inflammation, cardiovascular are valued as medicinal plant because they have been disease, cancer, diabetes) and some aging processes found to be effective in the treatment of skin wounds, (Benedi et al., 2004; Cui et al., 2004; Manosro et al., burns and gastrointestinal diseases (Silva et al., 2005; 2005; Kizil et al., 2008). Nevertheless, human body has Unal et al., 2008). Among these species, Hypericum evolved protective mechanisms to maintain the balance perforatum, also known as St John’s Wort, has attracted between the production and elimination of ROS. These much interest in recent years as a potential anti- protective mechanisms involve several specialised depressant (Barnes et al., 2001; Kizil et al., 2008). enzymes such as superoxide dismutase (SOD) and Hypericum perforatum has also been found to have a glutathione peroxidase (GPX) as well as non-enzymatic good antioxidative activity in vitro, which may aid in antioxidants such as reduced glutathione (GSH), ascor- general good health (El-Sherbiny et al., 2003; Benedi bate (ASC), thiols and phenolic compounds (Wickens, et al., 2004; Silva et al., 2005). Therefore, H. perforatum 2001; Urso & Clarkson, 2003). But these protective is considered to be a promising source of natural mechanisms are often insufficient for the completion of antioxidants. ROS scavenging. Therefore, dietary supplementation of In Taiwan, two native Hypericum species, namely Hypericum sampsonii (common name Sampson’s St *Correspondent: Fax: +886 4 37078702; John’s Wort) and Hypericum japonicum (common name e-mail: [email protected] Japanese St John’s Wort), have been used in traditional doi:10.1111/j.1365-2621.2009.02066.x Ó 2009 The Authors. Journal compilation Ó 2009 Institute of Food Science and Technology 2250 Several antioxidative enzymes in Hypericum C. L. Chen et al. medicine for the treatments of external wounds, burns subjected to freeze- and hot air-drying treatments were and snake bites. However, there are so far no reports also collected in the same day. The harvested samples related to the antioxidative activity of these two Hyper- (including leaves and stems) were dried in a force icum species. Moreover, medicinal herbs are often dried draught oven (F53; WTE binder, Tuttlingen, Germany) and stored for long time before use in manufacturing at 60 °C for 48 h or freeze-dried with a freezer dryer various types of product, and the quality of dehydrated (Lyphlock 12; Labcono, Kansas City, USA) at )50 °C. medicinal samples is strongly affected by the drying The dried samples collected from the same experiment process (Ratti et al., 2007; Que et al., 2008). But little plot, with 10% moisture content on dry weight base, information concerning the effects of drying on the were pooled and ground through a 2 mm mesh grinder, quality of dehydrated Hypericum materials are avail- and were sealed in polyester bottles and stored at able. Compared to hot air-drying, freeze-drying is )20 °C for later chemical analyses. generally better in preserving the medical quality of medicinal plants during processing (Abascal et al., Determinations of antioxidants 2005). Thus, the major objective of this study was to determine the activities of several antioxidative enzymes The content of thiol was determined by a colorimetric and the content of various antioxidants in H. sampsonii assay based on procedures described by Chan & Wass- and H. japonicum. The recently introduced H. perfora- erman (1993) with some modifications. Briefly, 150 mg of tum (St John’s Wort) was also used as reference for materials were homogenised and extracted using 1.5 mL comparison. The influences of freeze-drying and hot air- of 0.2 m Tris–HCl (pH 9.5) that containing 8 m urea, drying on the antioxidative responses of H. perforatum 10 mm disodium 2-nitro-5-thiosulfobenzoate (NTSB)2) plants were also evaluated and compared. and 3 mm EDTA for 25 min, and then centrifuged at 13 600 g for 10 min. A 100 lL aliquot of tissue extract was added to 1 mL of 0.2 m Tris-HCl (containing 8 mm Materials and methods urea, 3 mm EDTA and 1% sodium dodecyl sulphate). The mixture was left to stand at room temperature under Chemicals dark for 10 min, and then centrifuged at 13 600 g for The Folin & Ciocalteu’s phenol reagent, 2,2¢-azobis(2- 10 min. Absorbance measurement was taken at 412 nm amidinopropane) hydrochloride (AAPH), 6-hydroxy- using a spectrophotometer, and cysteine-HCl was used in 2,5,7,8-tetramethyl-chroman-2 carboxylic acid (trolox) the construction of the standard curve. and other chemicals were obtained from Sigma (Sigma For glutathione determinations, 200 mg materials Co., St Louis, MO, USA). were homogenised in a cold mortar and pestle with 1.5 mL ice-cold 5% (w ⁄ v) sulfosalicyclic acid and centrifuged at 15 000 g for 20 min. The supernatants Plant materials were used for reduced glutathione (GSH) and oxidised Hypericum sampsonii (Sampson’s St John’s Wort), glutathione (GSSG) determinations (Smith, 1985). Total H. japonicum (Japanese St John’s Wort) and H. perfo- glutathione (GSH+GSSG) was determined by adding ratum (St John’s Wort) plants were grown on raised 0.1 mL potassium phosphate buffer (0.5 m, pH 7.5), two-row beds (1 m wide and 6 m long with 30 cm bed 0.1 mL NADPH (2 mm in sodium phosphate buffer) spacing), covered with silver-black polyethylene sheets, and 0.1 mL glutathione reductase (2.5 units) to 0.05 mL in the experiment farm of the Department of Agron- supernatant. The reaction was monitored by the rate of omy, National Chung Hsing University. The experi- change in absorbance at 412 nm. Total glutathione was mental design was a randomised complete block design calculated from a standard curve in which GSH with four replicates (i.e. four experiment plots). The equivalents present were plotted against the rate of plant spacing was 30 · 30 cm. Pre-plant fertilisers were change in absorbance at 412 nm. GSSG was determined )1 )1 applied at the rates of 100 kg N ha ,60kgP2O5 ha using the same procedure as for total glutathione after )1 and 100 kg K2Oha . removal of GSH from the sample by 2-vinylpyridine The harvest of aerial tissues was conducted on 80 days derivatisations. GSH was determined by substracting after planting when Hypericum plants were fully devel- GSSG from the total glutathione content. oped in the field. Four randomly selected plants with For ASC and dehydroascorbate (DHA) determina- same size in each plot were manually harvested by tions, a modification of the method of Law et al. (1983) cutting the upper 15–16 cm of each shoot on the plant. was used. About 200 mg materials were homogenised in a The harvested shoots were wrapped into pre-wet cold mortar and pestle with 1.5 mL ice-cold 5% (v ⁄ v) moisten paper and stored in icebox to prevent wilting trichloracetic acid solution and centrifuged at 18 000 g during transfer to the laboratory. Once in the labora- for 20 min, and 5 lL of supernatant were used for total tory, the samples were stored at )70 °C for later ASC and ASC determinations. Total ASC was deter- analysis. Ten more H. perforatum plants that were mined by adding 5 lL sodium phosphate buffer (100 mm, International Journal of Food Science and Technology 2009 Ó 2009 The Authors. Journal compilation Ó 2009 Institute of Food Science and Technology Several antioxidative enzymes in Hypericum C.
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