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

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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 2009 The Authors. Journal compilation 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 2009 The Authors. Journal compilation 2009 Institute of Food Science and Technology Several antioxidative enzymes in Hypericum C. L. Chen et al. 2251 pH 7.4), 2.5 lL dithiothreitol (10 mm), 2.5 lL 0.5% 5% water soaking or 10% water extract was prepared N-ethylmaleimide, 10 lL 10% trichloracetic acid, 10 lL by extraction of various materials with de-ionised water, 44% H3PO4,10lL 4% bipyridyl (in 70% ethanol) and by soaking for 24 h or shaking for 2 h at room 5 lL 13% FeCl3 to 5 lL of supernatant. The reaction was temperature, respectively, and then centrifuged at monitored by the rate of change in absorbance at 525 nm. 12 000 g at 4 C. The antioxidant activity was deter- Total ASC was calculated from a standard curve in which mined by adding 100 lL sample to 850 lL phosphate ASC equivalents present were plotted against the rate of buffer (75 mm, pH7.0) and 150 lL phycoerythrin change in absorbance at 525 nm. ASC was determined (68 mg L)1) and 150 lL AAPH (50 mm). Absorbance using the same procedure as for total ASC, but without measurement was taken (emission 565 nm, excitation the addition of dithiothreitol and N-ethylmaleimide. 540 nm) using a fluorescence spectrophotometer (F2000; DHA content was then deduced from the difference Hitachi, Tokyo, Japan), and Trolox was used in the between total ASC and ASC contents. construction of the standard curve. The content of phenolics was estimated by a colorimetric assay based on procedures described by Taga et Statistical analysis al. (1984). About 200 mg of materials were homogenised in a cold mortar and pestle using 3 mL of 60% (v ⁄ v) The data were expressed as the mean of four replicate methanol containing 0.3% (v ⁄ v) HCl and left to stand determinations and standard deviation (SD). Statistical for 60 min, and then centrifuged at 18 000 g for 15 min. comparisons were made with Duncan’s multiple range A10lL aliquot of tissue extract was dissolved in test, based on the 0.05 significance level, for each of the 200 lLof2%(v⁄ v) Na2CO3, and 10 lL of the Folin & significant variables. Ciocalteu’s phenol reagent (50%, v ⁄ v) were added. The mixture was left to stand at room temperature for Results and discussion 30 min. Absorbance measurement was taken at 725 nm using a spectrophotometer, and caffeic acid was used in The contents of antioxidants the construction of the standard curve. The alcoholic extracts of H. perforatum (St John’s Wort), which contain many flavonoids and phenolics, have been Determinations of antioxidative enzyme activities demonstrated an antioxidative action (El-Sherbiny et al., For enzyme activity measurements, four replicates of 2003; Benedi et al., 2004; Silva et al., 2005). However, the materials were hand-ground at 4 C in a mortar and information on the levels of various antioxidants in pestle with various buffer solutions, and then centri- H. perforatum raw materials has been somewhat fuged. The supernatants were used for determination of neglected. Moreover, there are so far no reports related enzyme activity. SOD (SOD, EC 1.15.1.1) activity was to the antioxidative activity of H. japonicum (Japanese St assayed following the decrease in absorbance due to John’s Wort) and H. sampsonii (Sampson’s St John’s NADH oxidation (Paoletti et al., 1986). The SOD Wort). The contents of several antioxidants in H. japon- activity was expressed as the units of enzyme activity icum, H. sampsonii and H. perforatum were shown in needed to inhibit the reaction by 50% per gram on fresh Table 1. Thiol, which has redox-active sulfhydryl, is the weight per minute. GPX (GPX, EC 1.11.1.9) activity most important antioxidant capable of protecting living was assayed by following the changes of NADPH at cells from any kind of oxidative damage (Demirkol et al., 340 nm, which was detailed by Flohe & Gunzler (1984). 2004). The level of thiol in fresh tissues differed signifi- GPX activity was expressed as the changes of lmol of cantly among the tested Hypericum species (Table 1). The NADPH per gram on fresh weight per minute. Perox- level of thiol in fresh tissues of H. sampsonii was idase (POD, EC 1.11.1.7) activity was measured using 25.70 lmol per gram on fresh weight, which was consid- the procedures detailed by MacAdam et al. (1992). The erably higher than that of H. perforatum and H. japonicum activity was expressed as the production of tetraguaiacol (Table 1). All the tested Hypericum species contained per gram on fresh weight per minute. Ascorbate more thiol than that in vegetables such as asparagus peroxidase (APX, EC 1.11.1.11) activities were deter- (0.53 lmol per gram on fresh weight), red pepper mined using the methods of Nakano & Asada (1981). (0.42 lmol per gram on fresh weight), green bean The activity of APX was expressed as the production of (0.32 lmol per gram on fresh weight) or fruits such as ASC per gram on fresh weight per minute. papaya (0.19 lmol per gram on fresh weight) and strawberry (0.10 lmol per gram on fresh weight) reported by Demirkol et al. (2004). Determination of oxygen radical absorbance capacity The level of GSH, the central to defence mechanism Total antioxidant activity assay based on the reduction against intra- and extra-cellular oxidative stress, also of AAPH radical by antioxidants present in sample varied considerably among the tested Hypericum species extract was used in this study (Cao & Prior, 1999). The (Table 1). The highest levels of GSH and total glutathione

2009 The Authors. Journal compilation 2009 Institute of Food Science and Technology International Journal of Food Science and Technology 2009 2252 Several antioxidative enzymes in Hypericum C. L. Chen et al.

Table 1 The contents of thiol, total glutathione, reduced glutathione Table 2 The activities of uperoxide dismutase (SOD), glutathione (GSH), oxidised glutathione (GSSG), total ascorbate, ascorbate peroxidase (GPX), peroxidase (POD) and ascorbate peroxidase (ASC), dehydroascorbate (DHA) and phenolics, expressed on fresh (APX), expressed on fresh weight (FW) base, in fresh tissue of aerial weight (FW) base, in fresh tissue of aerial parts of field-grown parts of field-grown Hypericum perforatum, Hypericum sampsonii and Hypericum perforatum, Hypericum sampsonii and Hypericum japoni- Hypericum japonicum plants cum plants Hypericum Hypericum Hypericum Hypericum Hypericum Hypericum Trait perforatum sampsonii japonicum Trait perforatum sampsonii japonicum SOD 5.50 ± 0.77b mmol min–1 g–1FW 8.74 ± 0.29a Thiol 17.84 ± 1.02b lmol g)1 FW 7.34 ± 0.28c 8.60 ± 0.56a 25.70 ± 1.32a GPX 55.78 ± 0.85b nmol min–1 g–1 FW 71.35 ± 2.09a GSH 0.15 ± 0.02a 0.08 ± 0.01c 0.09 ± 0.01b 70.35 ± 2.03a GSSG 0.27 ± 0.02a 0.14 ± 0.01b 0.29 ± 0.01a POD 101.94 ± 5.24a 61.83 ± 0.87c 69.58 ± 2.62b Total 0.42 ± 0.01a 0.22 ± 0.01c 0.38 ± 0.01b APX 601.61 ± 4.60a 360.75 ± 3.07c 552.37 ± 7.67b Glutathione ASC 11.49 ± 0.86b 20.28 ± 3.89a 7.82 ± 2.88c Values (expressed as mean ± standard deviation) followed by different DHA 3.40 ± 0.20b 14.05 ± 0.84a 1.72 ± 0.02c letters within each row indicate significant differences according to Total 14.89 ± 1.58b 34.33 ± 1.91a 9.54 ± 0.94c Duncan’s test (P < 0.05). Ascorbate ) Phenolics 28.17 ± 3.94b mg g 1 FW 24.90 ± 1.87c responsible for catalysing the conversion of superoxide a 32.11 ± 2.31 to elemental oxygen and hydrogen peroxide (Cui et al., Values (expressed as mean ± standard deviation) followed by different 2004). In our study, H. japonicum had the highest SOD letters within each row indicate significant differences according to activity, and then followed by H. sampsonii and Duncan’s test (P < 0.05). H. perforatum (Table 2). SOD-produced hydrogen peroxide is converted to simple water and oxygen by several PODs each using different reducing agents (Cui (GSH + GSSG) were found in H. perforatum, and et al., 2004). Our results indicated that H. japonicum followed by H. japonicum and H. sampsonii. The levels of had the highest GPX, and then followed by H. GSH and total glutathione for H. sampsonii were about sampsonii and H. perforatum (Table 2). On the contrary, one-half of that of H. perforatum (Table 1). Hypericum H. perforatum had the highest POD and APX activities, sampsonii also had the lowest oxidised glutathione and then followed by H. japonicum and H. sampsonii (GSSG) among the three Hypericum species. The contents (Table 2). The activities of SOD were considerably of GSH in all the tested Hypericum species were relatively higher than POX, GPX or APX. Attempts to supple- low in comparison to asparagus, spinach, avocado or ment with dietary delivery of antioxidative enzymes green bean (Demirkol et al., 2004). These results indicate such as SOD or GPX naturally present in various plant- that some biothiols such as cysteine and its derivatives derived products prove disappointing, because these should also be studied in Hypericum tissues. enzymes are easily deactivated or destroyed by harsh Ascorbate is another antioxidant that play an impor- acids and enzymes contained in the digestive organisms tant role in protecting the cell against damage caused by of human body (Vouldoukis et al., 2004). Nevertheless, ROS (Doblado et al., 2005). Our study found that recent studies have indicated that supplementation with H. sampsonii had the highest levels of ASC, DHA and gliadin-combined standard plant SOD extract promotes total ASC (ASC + DHA), 20.28, 14.05 and 34.33 lmol the cellular antioxidant status and protects against per gram on fresh weight, respectively, among three oxidative stress-induced cell death (Muth et al., 2004). Hypericum species (Table 1). Hypericum japonicum had Our study found that the activities of SOD in the the lowest ASC, DHA and total ASC levels. examined Hypericum species were comparable to that of Hypericum genus is considered to be a rich source of melon extract rich in SOD (Vouldoukis et al., 2004). polyphenol that play an important role in inhibiting lipid Therefore, SOD in the aerial tissues of examined peroxidation (Silva et al., 2005; Kizil et al., 2008). In the Hypericum species might also be used as a vegetal current study, significant variations in the level of total antioxidant supplementation, provided that a suitable polyphenol also existed among the tested Hypericum protective technique against digestive process is species; with H. sampsonii contained more total polyphe- adopted. nol than H. perforatum and H. japonicum (Table 1). The effects of drying methods on antioxidant parameters The activities of antioxidative enzymes Medicinal herbs generally contain about 80% water that The activities of several antioxidative enzymes were needs to be dried to less than 15% before use in reported in Table 2. Superoxide dismutase (SOD) is manufacturing various types of product. Drying of

Table 3 The contents of thiol, reduced glutathione (GSH), oxidised Table 4 The activities of superoxide dismutase (SOD), glutathione glutathione (GSSG), total glutathione (GSH + GSSG), ascorbate peroxidase (GPX), peroxidase (POD) and ascorbate peroxidase (ASC), dehydroascorbate (DHA), total ascorbate (ASC + DHA) and (APX), expressed on dry weight base, in aerial part of field-grown phenolics, expressed on dry weight base, in aerial part of field-grown Hypericum perforatum plants subjected to different drying treatments Hypericum perforatum plants subjected to different drying treatments Hot Hot Trait Fresh Freeze-drying air-drying Trait Fresh Freeze-drying air-drying SOD 8.72 ± 0.89a (mmol min–1 g–1 DW) 5.42 ± 0.18b 1.34 ± 0.15c Thiol 118.98 ± 5.30c lmol g–1 DW 208.70 ± 12.90a GPX 0.37 ± 0.01a (lmol min–1 g–1 DW) 0.23 ± 0.08b 0.14 ± 0.01c 174.84 ± 5.75b POD 0.68 ± 0.05a 0.48 ± 0.04b 0.02 ± 0.01c GSH 0.96 ± 0.08a 0.97 ± 0.13a 0.12 ± 0.03b APX 4.01 ± 0.07a 2.65 ± 0.23b 0.19 ± 0.02c GSSG 1.83 ± 0.09a 0.91 ± 0.11b 0.27 ± 0.05c Total 2.79 ± 0.09a 1.87 ± 0.11b 0.39 ± 0.05c Values (expressed as mean ± standard deviation) followed by different Glutathione letters within each row indicate significant differences according to ASC 76.57 ± 4.31a 70.16 ± 12.04b 53.46 ± 7.27c Duncan’s test (P < 0.05). DHA 22.69 ± 0.47b 46.96 ± 11.05a 41.28 ± 3.32a Total 99.26 ± 10.55b 117.12 ± 12.04a 94.74 ± 5.27b Ascorbate treatments gave significant increase in DHA relative to Phenolics 187.77 ± 15.35b mg g–1 DW 352.82 ± 20.16a fresh material (Table 3). As a result, the increase in total 194.08 ± 15.82b ASC was large in freeze-dried material, while an

Values (expressed as mean ± standard deviation) followed by different insignificant decrease in total ASC was found for hot letters within each row indicate significant differences according to air-dried material (Table 3). Duncan’s test (P < 0.05). The content of phenolics in freeze-dried H. perforatum materials was slightly higher than that in fresh materials but the increase did not reach 5% significant level medicinal herbs is known to inhibit microorganism (Table 3). However, the content of phenolics in hot air- growth and forestall certain biochemical changes. But dried material was 1.9 times high than that in fresh drying can also cause changes in the physical properties material, indicating the formation of polyphenol during such as colour, structure and the deterioration of aroma hot air-drying. The formation of phenolic compounds compounds or degradation of nutritional substance might be due to the availability of precursors of phenolic (Ratti et al., 2007; Que et al., 2008). Hot air-drying is molecules by non-enzymatic interconversion between commonly used in drying process, but usually results in phenolic molecules (Que et al., 2008). inferior product quality. Freeze-drying is believed to be The method of drying also had significant effects on better in protecting nutrients during processing, but it is retention of antioxidative enzyme activity (Table 4). costly. As was shown in Table 3, the levels of total thiol Freeze-drying is reported to extensively perturb the in H. perforatum materials changed with drying meth- structure and diminish the activity and physical stability ods. Both freeze-drying and hot air-drying gave consid- of various proteins (Griebenow & Klibanov, 1995). Our erable increases in total thiol relative to fresh material study found that the freeze-dried H. perforatum material (Table 3). GSH, GSSG and total glutathione contents in retained an average of 65% of SOD, GPX, POD and fresh and dried H. perforatum materials also differed APX activities, relative to their fresh counterparts. The significantly (Table 3). Fresh aerial tissues of H. perfo- percentages of recovery in activity were 38% and 15% ratum had 0.96 lmol GSH per gram on dry weight base. for SOD and APX in hot air-dried H. perforatum Freeze-drying did not reduce the level of GSH relative to materials, respectively (Table 4). POD and APX activ- fresh material, whereas hot air drying did (Table 3). ities were especially diminished (less than 5% of Both drying treatments gave significant decreases in recovery in activity) following hot air-drying of GSSG relative to fresh material (Table 3). As a result, H. perforatum materials (Table 4). Both dehydration the decrease in total glutathione (GSH + GSSG) was and heat during drying process have shown capacity to about 33% in freeze-dried material and 86% in hot air- denature proteins (Tzannis & Prestreiski, 1999; França dried material (Table 3). et al., 2007). Thus, it appears that the tested ROS- There were considerable differences between fresh and scavenging enzymes might suffer proteolysis during dried materials in ASC level (Table 3). Freeze-drying freeze- or hot air-drying. did not significantly reduce the level of ASC relative to It is generally agreed that liquid form of dietary fresh material. Compared with freeze drying, hot air- supplement, either in ethanol or water extract, is best for drying greatly reduced the level of ASC (Table 3). These maximum absorption. A number of assays have been results are in agreement with the reports of Abascal used to measure the antioxidative activity of dietary et al. (2005) and Goula & Adamopoulos (2006). plant extracts. The ethanol extract of H. perforatum has Contrasting with the results of ASC, both drying been reported to show relevant antioxidant activity both

2009 The Authors. Journal compilation 2009 Institute of Food Science and Technology International Journal of Food Science and Technology 2009 2254 Several antioxidative enzymes in Hypericum C. L. Chen et al.

) Table 5 Oxygen radical absorbance capacity (ORAC) values, expres- sage (Salvia oﬃcinalis) (13.28 lmol Trolox eq. g 1 FW) sed on dry weight base, in aerial parts of Hypericum perforatum tissues or garden thyme (Thymus vulgaris) (19.49 lmol Trolox subjected to various drying and extraction treatments eq. g)1 FW) (Zheng & Wang, 2001). These results conﬁrm the superiority of H. perforatum in having good ORAC value )1 antioxidative activity in vitro. Treatment (lM Trolox eq. g DW) In conclusion, our study demonstrates that the 5% water soaking examined antioxidant traits vary considerably among Fresh Hypericum perforatum tissue 193.20 ± 13.89de the three tested Hypericum species. Hypericum sampsonii Freeze-dried 230.32 ± 16.54bc contains more thiol, phenolics and ASC than Hypericum perforatum tissue f H. perforatum and H. japonicum. All three Hypericum Hot air-dried 151.04 ± 4.10 species have extremely higher SOD activity in compar- Hypericum perforatum tissue Black tea leaves 176.15 ± 3.04ef ison with other antioxidative enzymes, suggesting that Green tea leaves 192.38 ± 16.32de SOD can be used as an antioxidant. The activities of 10% water extraction POD and APX for H. perforatum are highest among the Fresh Hypericum perforatum tissue 312.71 ± 2.04a Hypericum species. Both freeze-drying and hot air- Freeze-dried 261.05 ± 2.89b drying increase the levels of total thiol and total Hypericum perforatum tissue polyphenol in H. perforatum materials. Drying treat- Hot air-dried 217.04 ± 26.55cd ments, particularly hot air-drying, produced H. perfo- Hypericum perforatum tissue ratum materials with lower GSH, GSSG and ASC. Both

Values (expressed as mean ± standard deviation) followed by different drying treatments decrease the activities of all the tested letters indicate significant differences according to Duncan’s test ROS-scavenging enzymes. Thus, the reduced amounts (P < 0.05). of GSH, GSSG and ASC as well as reduced activity of ROS scavenging enzymes might contribute to the lower ORAC in dried H. perforatum materials, particularly in in vitro and in a cell system (El-Sherbiny et al., 2003; hot air-dried materials. Benedi et al., 2004; Silva et al., 2005). However, information on the level of antioxidative activity in the References water extract of H. perforatum is poorly understood. This study, the oxygen radical absorbance capacity Abascal, K., Ganora, L. & Yarnell, E. (2005). The effect of freeze (ORAC) was determined in H. perforatum materials on drying and its implications for botanical medicines: a review. Phytotherapy Research, 19, 655–660. water base using the technique detailed by Cao & Prior Barnes, J., Anderson, L.A. & Phillipson, J.D. (2001). St John’s wort (1999). The ORAC values of 5% water soakings and (Hypericum perforatum L.): a review of its chemistry, pharmacology 10% water extractions prepared by fresh, freeze-dried and clinical properties. Journal of Pharmacy and Pharmacology, 53, and hot air-dried H. perforatum materials were reported 583–600. in Table 5. Green tea and black tea are known to Benedi, J., Arroyo, R., Romero, C., Martıń-Aragoń, S. & Villar, A.M. (2004). Antioxidant properties and protective effects of a standard- contain flavanoid derivatives of catechins in significant ized extract of Hypericum perforatum on hydrogen peroxide-induced amounts and exhibit greater antioxidative activity (Ri- oxidative damage in PC12 cell. Life Science, 75, 1263–1276. chelle et al., 2001). Therefore, both green tea and black Cao, B.G. & Prior, R.L. (1999). Measurement of oxygen radical were also used as reference for comparison (Table 5). absorbance capacity in biological samples. Methods in Enzymology, 299, 50–62. Freeze-dried H. perforatum materials exhibit signifi- Chan, Y.K. & Wasserman, B. (1993). Direct colorimetric assay of free cantly higher ORAC than fresh materials by using 5% thiol groups and disulfide bounds in suspensions of solubilized and water soaking. However, hot air-drying materials exhib- particulate cereal proteins. Cereal Chemistry, 70, 22–26. ited considerably lower ORAC than fresh materials. On Cui, K., Luo, X., Xu, K. & Ven Murthy, M.R. (2004). Role of the contrary, both freeze-drying and hot air-drying oxidative stress in neurodegeneration: recent developments in assay methods for oxidative stress and nutraceutical antioxidants. Pro- showed significant decrease in ORAC relative to fresh gress in Neuro-Psychopharmacology and Biological Psychiatry, 28, materials when the antioxidative activities were deter- 771–799. mined on 10% water extraction (Table 5). Almost all Demirkol, O., Adams, C. & Ercal, N. (2004). Biologically important the H. perforatum materials (except hot air-dried mate- thiols in various vegetables and fruits. Journal of Agricultural Food Chemistry, 52, 8151–8154. rials subjected to 5% water soaking) showed greater Doblado, R., Zielinski, H., Piskula, M., et al. (2005). Effect of ORAC than black or green tea leaves (Table 5). Our processing on the antioxidant vitamins and antioxidant capacity of ORAC values are greater than the ORAC values Vigna sinensis var. Carilla. Journal of Agricultural Food Chemistry, detected from green pepper (160 lmol Trolox eq. g)1 53, 1215–1222. DW), purple onion (153 lmol Trolox eq. g)1 DW) or El-Sherbiny, D.A., Khalifa, A.E., Attia, A.S. & Eldenshary, E.E.S. )1 (2003). Hypericum perforatum extract demonstrates antioxidant broccoli (132 lmol Trolox eq. g DW) (Ou et al., 2002). properties against elevated rat brain oxidative status induced by Moreover, these ORAC values are also greater than the amnestic dose of scopolamine. Pharmacology Biochemistry Behavior, ORAC values found for medicinal plants such as garden 76, 525–533.

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2009 The Authors. Journal compilation 2009 Institute of Food Science and Technology International Journal of Food Science and Technology 2009