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Caffeic acid derivatives, total phenols, antioxidant and antimutagenic activities of Echinacea purpurea flower extracts
LWT - Food Science and Technology 46 (2012) 169e176
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Caffeic acid derivatives, total phenols, antioxidant and antimutagenic activities of Echinacea purpurea flower extracts Yu-Ling Tsai a, b, Shiow-Ying Chiou c, Kung-Chi Chan b, Jih-Min Sung c, Sheng-Dun Lin c, * a
Department of Nutrition, Hungkuang University, 34 Chung-Chie Road, Shalu, Taichung 43302, Taiwan, ROC Department of Food and Nutrition, Providence University, 200 Chung-Chie Road, Shalu, Taichung 43301, Taiwan, ROC c Department of Food Science and Technology, Hungkuang University, 34 Chung-Chie Road, Shalu, Taichung 43302, Taiwan, ROC b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 May 2011 Received in revised form 27 July 2011 Accepted 30 September 2011
This study evaluated the effects of ethanol concentration and extraction temperature on the quality of extracts obtained from freeze-dried Echinacea purpurea flowers. The antioxidant and antimutagenic effects of flower extract were also examined. The optimal extraction conditions for freeze-dried flowers were 50% aqueous ethanol and 65 C extraction temperature, with extraction yield of 37.4%. The contents of total phenols, caffeic acid derivatives, and cichoric acid in freeze-dried extracts were 473.34 mg chlorogenic acid equivalents/g, 302.20 mg/g and 217.61 mg/g, respectively. The 50% ethanolic flower extract did not show toxicity and mutagenicity toward Salmonella typhimurium TA98 and TA100 with or without S9 mix. The ethanolic extract at 0.25e5 mg/plate exhibits a dose-dependent inhibitory effect against the mutagenicity of 2-aminoanthracene. Thus, freeze-dried E. purpurea flower ethanolic extract exhibits good antioxidant and antimutaginic activities. Ó 2011 Elsevier Ltd. All rights reserved.
Keywords: Echinacea purpurea Caffeic acid derivative Antioxidant Antimutagenicity
1. Introduction Echinacea purpurea L. is one of the top selling medicinal plants widely used to alleviate colds, sore throats and other upper respiratory infections. The prepared Echinacea products (e.g., infusions, tinctures and capsules) are used to stimulate immune system, and their immuno-stimulating properties are attributed to the bioactive phytochemicals including caffeic acid derivatives, alkamides, polysaccharides, and glycoproteins. Among these phytochemicals, caffeic acid derivatives, especially cichoric acid, possess many bioactive functions including anti-hyaluronidase activity, protection of collagen from free radical induced degradation, antiviral activity, inhibition of human immunodeficiency virus type 1 integrase and replication, promoting phagocyte activity in vitro and in vivo and a high free radical scavenging property (Barnes, Anderson, Gibbons, & Phillipson, 2005; Bauer, & Wagner, 1991; Pellati, Benvenuti, Magro, Melegari, & Soragni, 2004). Thus, cichoric acid is commonly used as marker to determine the medicinal quality of Echinacea products (Thygesen, Thulinn, Mortensen, Skibsted, & Molgaard, 2007). Various extraction techniques have been developed to obtain phytochemicals from plant materials (Wang & Weller, 2006). Both
* Corresponding author. Tel.: þ886 4 2631 8652x5038; fax: þ886 4 2631 9176. E-mail address: [email protected] (S.-D. Lin). 0023-6438/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.lwt.2011.09.026
the classical extraction methods including solvent extraction with or without mechanical agitation (or shaking) and some novel techniques such as ultrasound-assisted extraction and microwaveassisted extraction are fast and efficient for extracting phytochemicals from plant materials. The ultrasound-assisted extraction and microwave-assisted extraction have many advantages over classical extraction methods in terms of extraction time, solvent consumption, extraction yields and reproducibility. However, these novel techniques have only been tested in a limited field of applications (Wang & Weller, 2006). Nevertheless, from industrial-scale production viewpoint, the selected extraction technique must be versatile, simple, low-cost and safe for both the operating personnel and the consumers (Wu, Murthy, Hahn, Lee, & Paek, 2008). In Taiwan, the classical solvent extraction coupled with higher temperature has been commercially used to obtain bioactive compounds from various plant species for years. Type of solvent is an important parameter affecting the recovery of phytochemicals during extractions (Wang & Weller, 2006). Alcoholic solvents, such as ethanol and methanol at different concentrations in water, have been used to extract phenols from plant materials (Alothman, Bhat, & Karim, 2009). Pellati et al. (2004) found that methanol/water extraction was more efficient than ethanol/water extraction for extracting total phenols in Echinacea. However, ethanol is non-toxic, it is easy to be recycled and mixed with water in different ratios. Besides, the acceptability of ethanol and water for human consumption models is high.
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Extraction temperature is another important parameter needs to be optimized during extraction (Camel, 2000; Kim, Murthy, Hahn, Lee, & Paek, 2007; Stuart & Wills, 2000; Wang & Weller, 2006). An increase in the working temperature generally favors extraction on both the solubility of solute and the diffusion coefficient, but also that beyond a certain value phenolic compounds can be denatured (Spigno, & De Faveri, 2007). E. purpurea has recently been introduced into Taiwan and grows well, and its flower was recently permitted for use as a food material by the Taiwan government (Taiwan Food and Drug Administration, 2011). The total phenols and caffeic acid derivatives contents in different plant parts were in the descending order: flowers > leaves > stems > roots (Lin, Sung, & Chen, 2011). However, little information is available regarding the phytochemicals, antioxidant and antimutagenic activities of E. purpurea flower extracts. Therefore, the objectives of this study were to determine the effects of various concentrations of ethanol and extraction temperatures on the extraction yields, total phenols, and caffeic acid derivatives of freeze-dried E. purpurea flowers, and to investigate the antioxidant properties of the ethanolic extract of E. purpurea flowers. The antimutagenic effects of E. purpurea flower extract by using Salmonella typhimurium test (Ames test) were also determined. 2. Materials and methods 2.1. Materials The flowers of E. purpurea (L.) Moench variety CLS-P2 harvested from six-month-old plants were donated by Echili Biotechnology (Taichung, Taiwan). The flowers were freeze-dried with vacuum on the same day as harvested. The dried flowers were ground in a mill, and screened through a 0.5 mm sieve. The dried flower powders were stored in sealed PET/Al/PE bag for 9 months at 20 C. Methanol, acetonitrile and phosphoric acid were purchased from Mallinckrodt Baker, Inc. (New Jersey, USA). Caftaric acid, echinacoside, cichoric acid and cynarin were purchased from ChromaDex Inc. (Santa Ana, CA, USA). Chlorogenic acid, sodium tungstate dehydrate, phosphomolybdic acid hydrate 1,1-diphenyl2-picrylhydrazyl (DPPH), trichloroacetic acid, potassium ferricyanide, ferrous chloride, ferrozine, ascorbic acid, a-tocopherol, butylated hydroxyanisole (BHA), ethylenediaminetetraacetic acid (EDTA), 2-aminoanthracene (2-AA), biotin, dimethyl sulfoxide (DMSO), glucose-6-phosphate (G-6-P), histidine, 4-nitro-o-phenylenediamine (4-NP) and sodium azide (NaN3) were purchased from SigmaeAldrich Chemical Co. (St. Louis, MO, USA). Anhydrous sodium carbonate was purchased from Shimakyu’s Pure Chemicals (Osaka, Japan). Sodium phosphate and ferric chloride were purchased from Wako Pure Chemical Industries Co. (Osaka, Japan). A metabolic activation fraction (S9 mix) prepared from SpragueeDawley rat liver was purchased from Nature Opera Biotechnology Inc. (Taipei, Taiwan). Ethanol (95%) was purchased from Taiwan Tobacco & Liquor Co. (Tainan, Taiwan). 2.2. Ethanol extracts preparation 2.2.1. Effect of various concentrations of ethanol The reverse osmosis (RO) water was prepared from tap water by RO machine system (Lepa Industrial Co., Ltd., Taipei, Taiwan). The 25%, 50%, and 75% aqueous ethanol were prepared from 95% ethanol mixed with RO water. The ethanolic extracts of E. purpurea flowers were obtained by extracting the ground flower powder (10 g) with 100 ml of RO water, 25%, 50%, 75%, and 95% aqueous ethanol in a shaking bath (25 C) (SB302, Kansin Instruments Co., Kaohsiung, Taiwan) at 100 rpm for 30 min, and then centrifuged at
3460g for 10 min, filtered through Whatman No.1 filter paper. The residue was re-extracted with two extra 100 ml portions of solvent as described above. The combined ethanolic extracts were rotary evaporated at 40 C and then freeze-dried with vacuum. Dry extracts thus obtained were stored at 20 C before use. The extracts extracted with 0%, 25%, 50%, 75%, and 95% ethanol were designated as E0, E25, E50, E75, and E95, respectively. All treatments were randomly produced. All experiments were done in triplicate. 2.2.2. Effect of various extraction temperatures The flower powders (10 g for each sample) were extracted with 100 ml of 50% aqueous ethanol in a shaking bath at 100 rpm for 30 min under 25 C, 35 C, 45 C, 55 C, 65 C, 75 C, 85 C, or 95 C conditions, and then centrifuged at 3460 g for 10 min, filtered through Whatman No.1 filter paper. The residue was re-extracted with two extra 100 ml portions of solvent as described above. The combined ethanolic extracts were rotary evaporated at 40 C and then freeze-dried with vacuum. Dry extracts thus obtained were stored at 20 C before use. The extracts prepared under 25 C, 35 C, 45 C, 55 C, 65 C, 75 C, 85 C, or 95 C conditions were designated as ET25, ET35, ET45, ET55, ET65, ET75, ET85, and ET95, respectively. All experiments were done in triplicate. 2.3. Determination of total phenols The content of total phenols in the extracts was determined using the Folin-Denis method (Perry, Burgess, & Glennie, 2001) with minor modifications. Folin-Denis reagent was prepared according to the method of Perry et al. (2001). Each extract (100 mg) was dissolved in 70% ethanol (4.0 ml) using an ultrasonic bath with 40 kHz for 1 min, and then the volume was adjusted to 5 ml. FolineDenis reagent (2.5 ml) was added to either 0.25 ml of extract or standard solution. After 3 min 35% sodium carbonate solution (2.5 ml) was added and the test solution was made up to 25 ml with H2O and mixed. After 45 min of incubation in room temperature, the test solution was centrifuged at 3460 g for 10 min. The absorption value was determined at 745 nm, with chlorogenic acid used as a standard. The total phenols were expressed as mg chlorogenic acid equivalents per g dry extract [the equation of standard curve: absorbance at 745 nm ¼ 0.5441 Cchlorogenic acid (mg/ ml) þ 0.0441, R2 ¼ 0.9963]. Each analysis was carried out in triplicate. 2.4. Determination of caffeic acid derivatives The content of caffeic acid derivatives in the extracts was determined according to the methods of Hu and Kitts (2000) and Pellati et al. (2004) with minor modifications. Briefly, each extract (100 mg) was dissolved in 70% ethanol (4.0 ml) using an ultrasonic bath with 40 kHz for 1 min, and then the volume was adjusted to 5 ml. The solution was filtered using a PVDF syringe filter (13 mm 0.45 mm) prior to injection onto an HPLC. The HPLC system consisted of a Hitachi L-2130 pump, a Hitachi L-2400 UV detector, and a LiChrospherÒ 100 RP-18e column (250 mm 4.6 mm 5 mm, Merck Co., Germany). The mobile phase was (A) acetonitrile/water containing 0.1% H3PO4 (10:90) and (B) acetonitrile/water containing 0.1% H3PO4 (25:75). A gradient elution profile was used with B increasing from 0% to 100% in 30 min and maintained at 100% for 10 min. Then a linear gradient of 100% B decreased to 0% B in 10 min. The flow rate was 1.5 ml/min, and the wavelength of the UVevisible detector was set at 330 nm. The sample injection volume was 20 ml. Contents of various caffeic acid derivatives were calculated on the basis of the calibration
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Table 1 Extraction of freeze-dried E. purpurea flowers with various concentrations of ethanol at 25 C: Extraction yield, content of total phenolics and caffeic acid derivatives in dry extracts. Extraction method
Extraction yield (%)a
E0 E25 E50 E75 E95
39.8 36.9 34.3 28.0 3.3
1.3Ab 0.9B 0.1C 1.2D 0.6E
Content (mg/g extract) Caftaric acid
Chlorogenic acid
Echinacoside
16.56 42.71 67.07 50.04 16.35
0.14 0.68 2.41 2.84 0.53
0.53 0.79 1.02 1.07 0.54
0.23D 3.86C 0.67A 1.39B 0.27D
0.01C 0.15B 0.01A 0.10A 0.02B
0.06C 0.03B 0.09A 0.07A 0.04C
Cichoric acid 37.23 108.86 180.29 160.95 42.54
0.25D 4.61C 1.76A 5.12B 0.36D
Cynarin
Total caffeic acid derivatives
nd nd nd nd nd
54.46 153.04 250.79 214.90 59.96
0.19D 4.33C 2.42A 5.12B 0.41D
Total phenolics 97.07 282.78 441.33 399.82 104.47
6.91D 6.44C 3.54A 8.37B 3.80D
Means with different capital letters in the same column are significantly different (P < 0.05). nd: not detected. a Extraction yield (%) ¼ (dried extract weight/sample weight) 100%. b Each value is expressed as mean SD (n ¼ 3).
curve of each caftaric acid, chlorogenic acid, echinacoside, cichoric acid and cynarin. Each analysis was carried out in triplicate. 2.5. Antioxidant properties 2.5.1. Scavenging ability on 1,1-diphenyl-2-picrylhydrazyl radicals For DPPH radicals scavenging ability assay (Shimada, Fujikawa, Yahara, & Nakamura, 1992), each extract (10e50 mg/ml, 4 ml) in methanol was mixed with 1 ml of methanolic solution containing DPPH radicals, resulting in a final concentration of 0.2 mM DPPH. The mixture was shaken vigorously and left to stand for 30 min in the dark, and the absorbance of the mixture was then measured at 517 nm against a blank. Ascorbic acid, BHA, and a-tocopherol were used for comparison. 2.5.2. Reducing power The reducing power was determined according to the method of Oyaizu (1986). Each extract (100e500 mg/ml, 2.5 ml) in methanol was mixed with 2.5 ml of 200 mM sodium phosphate buffer (pH 6.6) and 2.5 ml of 1% potassium ferricyanide, and the mixture was incubated at 50 C for 20 min. After 2.5 ml of 10% trichloroacetic acid (w/v) were added, the mixture was centrifuged at 2600 g for 10 min. The upper layer (5 ml) was mixed with 5 ml of deionized water and 1 ml of 0.1% ferric chloride, and the absorbance of the mixture was measured at 700 nm against a blank to determine the amount of ferric ferrocyanide (Prussian blue) formed. Ascorbic acid, BHA, and a-tocopherol were used for comparison. 2.5.3. Ferrous ions chelating abilities Chelating ability was determined according to the method of Dinis, Madeira, and Almeida (1994). Each extract (1e5 mg/ml, 1 ml)
in methanol was mixed with 3.7 ml of methanol and 0.1 ml of 2 mM ferrous chloride. After 30 s of standing, 0.2 ml of 5 mM ferrozine was added. After 10 min at room temperature, the absorbance of the mixture was determined at 562 nm against a blank. The EDTA, ascorbic acid, BHA, and a-tocopherol were used for comparison. 2.6. Ames test 2.6.1. Salmonella strains The histidine-requiring strains of S. typhimurium TA 98 and TA 100 were purchased from the Bioresources Collection and Research Center (BCRC), Food Industry Research and Development Institute (FIRDI, Hsinchu, Taiwan). The test strains were characterized for spontaneous reversion and genetic analysis including histidine requirement (his-), UV (uvrB) sensitivity, crystal violet (rfa) sensitivity and ampicillin resistance (Mortelmans & Zeiger, 2000). 2.6.2. Toxicity test For toxicity measurement (Waleh, Rapport, & Mortelmans, 1982), the 50% ethanolic extracts (0.1 ml) from freeze-dried E. purpurea flower (0e5 mg/plate), 0.5 ml of S9 mix or phosphate buffer saline (PBS, 0.1 M, pH 7.4) were mixed with 0.1 ml PBS and 0.1 ml of 12 h-cultured test strain (approximately 108 cells/ml). After incubation for 20 min at 37 C, the serial dilutions were made with nutrient broth no. 2 (OXOID), an aliquot (1 ml) was mixed with 15 ml of nutrient agar and incubated at 37 C for 48 h, and the number of colonies was counted. 2.6.3. Mutagenicity test Mutagenicity was assayed by the standard Ames test (standard plate incorporation assay) (Maron & Ames, 1983; Mortelmans &
Table 2 Extraction of freeze-dried E. purpurea flowers with 50% ethanol at various extraction temperatures: Extraction yield, content of total phenolics and caffeic acid derivatives in dry extracts. Extraction method
Extraction yield (%)a
ET25 ET35 ET45 ET55 ET65 ET75 ET85 ET95
34.3 34.4 35.7 36.4 37.4 38.2 39.0 39.8
0.1Gb 0.4G 0.5F 0.2E 0.6D 0.4C 0.3B 0.3A
Content (mg/g extract) Caftaric acid 67.07 67.96 72.13 72.79 78.18 75.59 74.06 72.43
0.67D 0.99D 1.83C 0.60BC 1.39A 1.11AB 1.42BC 0.66C
Chlorogenic acid 2.41 2.37 3.36 3.84 4.27 3.99 3.42 2.79
0.01E 0.03E 0.31C 0.07B 0.04A 0.12B 0.16C 0.09D
Echinacoside 1.02 1.53 2.16 2.03 2.14 1.98 1.83 1.65
0.09E 0.15D 0.20A 0.00A 0.05A 0.00AB 0.02BC 0.03CD
Means with different capital letters in the same column are significantly different (P < 0.05). nd: not detected. a Extraction yield (%) ¼ (dried extract weight/sample weight) 100%. b Each value is expressed as mean SD (n ¼ 3).
Cichoric acid 180.29 182.98 191.22 191.86 217.61 205.99 196.49 186.35
1.76E 3.95E 1.86CD 1.19CD 4.50A 2.20B 2.54C 4.39DE
Cynarin
Total caffeic acid derivatives
Total phenolics
nd nd nd nd nd nd nd nd
250.79 2.42E 254.84 3.07E 268.87 3.58CD 270.52 1.72CD 302.20 5.98A 287.55 0.97B 275.80 3.83C 263.22 5.17D
441.33 463.00 464.50 466.92 473.34 458.02 456.16 434.41
3.54E 3.54BCD 3.30BC 2.71AB 2.42A 1.73CD 3.77D 3.08E
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2.6.4. Antimutagenicity assay For the antimutagenicity test, the 50% ethanolic extract from freeze-dried E. purpurea flower and one of three mutagens for each strain were assayed. The positive controls using 4-NP (1.25 mg/ plate) for TA98 strain and NaN3 (1.25 mg/plate) for TA100 strain were tested without S9 metabolic activation whereas those using 2-AA for both TA98 (0.25 mg/plate) and TA 100 (2 mg/plate) strains was tested with S9 activation. The mutagen in PBS (direct test) or S9 mix (indirect test) was added to bacterial culture with the ethanolic extract. After incubation for 20 min at 37 C, the mixture was added to 2.0 ml of top agar and plated as described above. Positive and negative controls were also conducted in each assay. The mutagenicity of appropriate mutagen for each strain in the absence of test samples was defined as 0% inhibition. The % inhibition was calculated as follows according to the method of Ong, Wong, Stewart, and Brockman (1986): % inhibition ¼ [1 (T S)/(M S)] 100, where T is the number of revertants per plate in the presence of mutagen and the test sample, M is the number of revertants per plate in the positive control and S is the number of spontaneous revertants per plate. The inhibition of 25e40% was considered as moderate antimutagenic effect and that of more than 40% was the strong effect. However, the inhibition of less than 25% were considered as weak and was not recognized as a positive result (Ikken et al., 1999).
3. Results and discussion 3.1. Effect of various concentrations of ethanol on the extraction yields, total phenols and caffeic acid derivatives The extraction yield is a measure of the solvent efficiency to extract specific components from the original materials. In the case of E. purpurea, it will give an idea about the extractability of total phenolics including the caffeic acid derivatives under different extraction conditions. Stanisavljevi c, Stojicevi c, Veli ckovi c, Veljkovi c, and Lazi c (2009) used both classical solvent extraction and ultrasound-assisted extraction to investigate the extraction yield of total phenols in aerial part of E. purpurea L., and found that the extract obtained by classical solvent extraction contained more phytochemicals and had greater antioxidant activities than the
80
60
40
20
0 0
10
20
30
40
50
400
500
4
5
Concentration (µ µg/ml)
B
2.5 2.0 1.5 1.0 0.5 0.0 0
100
200
300
Concentration (µ µg/ml)
100
2.7. Statistical analysis
C
80 Chelating ability (% )
All measurements were carried out in triplicate. All data were subjected to analysis of variance using the Statistical Analysis System software package (SAS Institute, Cary, NC, USA). When a significant difference was found among treatments, Duncan’s multiple range tests were performed to determine the differences among the mean values at the level of a ¼ 0.05.
A
100
Scavenging ability (% )
Zeiger, 2000). Both TA98 and TA100 strains were aerobically grown at 37 C in nutrient broth no. 2 (OXOID) supplemented with ampicillin. An aliquot (0.1 ml) of the overnight incubated bacterial culture was mixed with ethanolic extracts (0.25e5 mg/plate) and 0.5 ml PBS (0.1 M, pH 7.4). After incubation for 20 min at 37 C, the mixture was added to 2.0 ml of top agar containing 0.05 mM biotinhistidine and plated onto glucose minimal agar plates (Mortelmans & Zeiger, 2000). For indirect test, 0.5 ml of S9 mix was supplemented instead of phosphate buffer. The activation system (S9 mix) contained 4% of Aroclor 1254-induced rat liver homogenate S9. The reversion rate was compared to that of control plates treated with 50% DMSO. Hisþ revertants were counted after incubation of the plates at 37 C for 48 h. Subsequently, the number of histidineindependent revertant colonies was numerated. Each assay was conducted in triplicate.
Absorbance at 700 nm
172
60
40
20
0 0
1
2
3
Concentration (mg/ml) Fig. 1. Antioxidant properties of E. purpurea flower extracts, ascorbic acid, butylated hydroxyanisole (BHA), a-tocopherol and ethylenediaminetetraacetic acid (EDTA). (A) scavenging ability on 1,1-dipheny1-2-picrylhydrazyl radicals. (B) reducing power. (C) ferrous ions chelating ability. Flower extract: The extract was prepared from vacuumdried E. purpurea flowers with 50% aqueous ethanol and 65 C extracting temperatures. Each value is expressed as mean standard deviation (n ¼ 3). -C-, E. purpurea extract; -7-, ascorbic acid; ---, a-tocopherol; -A-, BHA; -B-, ethylenediaminetetraacetic acid.
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E. purpurea flowers (Table 1). Therefore, only 50% ethanol was chosen for optimizing extraction temperature.
extract obtained by ultrasonic extraction. They concluded that the results were due to the degradation of total phenols by interaction with the highly reactive hydroxyl radicals formed during sonication. Therefore, in the present study, only the traditional solvent extraction was used to extract the phytochemicals in freeze-dried E. purpurea flowers. Table 1 shows that the extraction yields of freeze-dried E. purpurea flowers were significantly affected by the ethanol concentrations (P < 0.05), with water extraction giving the highest extraction yield. When the ethanol volume percentage in the solvent was increased up to 95%, the extraction yields were decreased from 39.8% to 3.3%. These results are related to solvent polarity and the solubility of components in tested sample (Xi et al., 2009). The contents of total phenols, individual and total caffeic acid derivatives in extracts were also affected by the ethanol concentrations (Table 1). A protocol of 70% ethanol extraction procedure has been proposed for the manufacture of E. purpurea ethanolic extracts (Spelman, Wetschler, & Cech, 2009). In this study, when the ethanol volume percentage was lower than 50%, the contents of total phenols, caftaric acid, chlorogenic acid, echinacoside, cichoric acid, and total caffeic acid derivatives were increased with the increases of ethanol concentrations. However, the total phenols, individual and total caffeic acid derivatives contents in extracts were decreasing when the ethanol concentration was higher than 50%. When the extraction yields were taken into consideration in this study, the total phenols, individual and total caffeic acid derivatives contents (mg/g freeze-dried flowers) of freeze-dried E. purpurea flowers are in the descending order of E50 > E75 y E25 > E0 > E95. As for the caffeic acid derivatives detectable in the freeze-dried flowers extracts (Table 1), cichoric acid (ranged from 68.36 to 74.90%) was the major content, followed by caftaric acid (ranged from 23.09 to 30.41%). This result is in agreement with the report of Zhai et al. (2007). Several studies (Stuart & Wills, 2000; Wu et al., 2008) have shown that cichoric acid and other active ingredients in E. purpurea root are optimally extracted with 60% ethanol. Similarly our results indicate that 50% ethanol is favorable for extracting individual caffeic acid derivatives in freeze-dried
3.2. Effect of various extraction temperatures on the extraction yields, total phenols and caffeic acid derivatives Table 2 shows that extraction yields of freeze-dried E. purpurea flowers ranged from 34.3% at 25 C to 39.8% at 95 C, with the highest extraction yield obtained from the ET95. The contents of total phenols, individual and total caffeic acid derivatives of 50% ethanol extracts were also greatly affected by the extraction temperatures. The total phenols, individual and total caffeic acid derivatives contents of extracts was enhanced by elevated extraction temperatures ranged from 25 C to 65 C. These increments in the active ingredients with the increase of temperature might be the result of an increased diffusivity of the solvent into the cells and an enhanced de-sorption of the components forming the cells (Camel, 2000; Wu et al., 2008). When the extraction yields were taken into consideration, the active ingredients contents of freezedried E. purpurea flowers also has noticeable increase with increasing extraction temperatures of 25e65 C. But the active ingredients contents of extracts showed a reverse trend between 65 C and 95 C of extraction temperatures. When the extraction yields were taken into consideration, total phenols and caftaric acid contents (mg/g freeze-dried flowers) change of flowers were not significantly (P > 0.05), total caffeic acid derivatives, cichoric acid, chlorogenic acid, and echinacoside contents in flowers showed a slightly decrease between 65 C and 95 C of extraction temperatures. In this study, the 65 C of extraction temperature gave higher total phenols, individual and total caffeic acid derivatives than the other extraction temperatures tested. Wu et al. (2008) reported that the treatment of the samples with 60% ethanol at 60 C was proven to be the most suitable procedure. 3.3. Antioxidant properties The DPPH radical scavenging ability of flower extract and standards are reported in Fig. 1A. The DPPH scavenging ability
Table 3 Toxicity and mutagenicity of ethanolic extract from freeze-dried E. purpurea flower toward Salmonella typhimurium TA98 and TA100 with and without S9 mix. Extract (mg/plate)
Survival (%)a TA98
TA100
S9
þS9
S9
(A) Toxicity 0.0 0.25 0.5 1.0 2.5 5.0
100.00Ab 86.19 5.47A 91.32 10.41A 86.44 6.74A 95.11 12.13A 96.72 6.57A
100.00B 97.28 107.76 106.21 115.62 97.77
Extract (mg/plate)
Hisþ revertants/plate (Mutagenicity ratio)c
7.02B 13.25AB 6.99AB 8.18A 4.28B
TA98
33 32 32 34 34 31
100.00B 99.37 108.20 105.47 99.40 91.80
3.51B 5.79A 5.95AB 2.64B 2.28C
100.00C 117.99 116.01 113.95 111.67 107.93
10.57A 0.46AB 5.05AB 1.01AB 1.88BC
TA100
S9 (B) Mutagenicity 0.0 0.25 0.5 1.0 2.5 5.0
þS9
þS9 3 3 4 6 4 7
(1.00)Ab (0.96)A (0.98)A (1.04)A (1.02)A (0.94)A
60 67 59 62 61 55
S9 7 (1.00)A 11 (1.12)A 4 (0.99)A 4 (1.04)A 4 (1.02)A 5 (0.93)A
Means with different capital letter within a column are significantly different (P < 0.05). a Survival (%) ¼ (number of colonies of extract per plate/number of colonies of control per plate) 100%. b Each value is expressed as mean standard deviation (n ¼ 3). c Mutagenicity ratio ¼ induced revertants per plate/spontaneous revertants (control) per plate.
142 140 147 135 141 156
þS9
7 (1.00)A 9 (0.99)A 7 (1.04)A 17 (0.95)A 19 (0.99)A 4 (1.10)A
125 104 113 129 120 125
5 (1.00)A 7 (0.83)A 10 (0.91)A 13 (1.03)A 1 (0.96)A 16 (1.00)A
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3.4. Toxicity, mutagenicity and antimutagenicity Many phenolic compounds were found to possess antimicrobial activity (Yam, Shah, & Hamilton-Miller, 1997). In this study, a good portion of total phenols and caffeic acid derivatives were present in the 50% ethanolic extracts of freeze-dried E. purpurea flower (Table 2). Therefore, a toxic concentration test preceding the antimutagenicity studies was performed. The result shows that the different concentrations (0.25, 0.5, 1.0, 2.5 and 5.0 mg/plate) of freeze-dried E. purpurea flower extract added to Ames indicator bacteria did not influence their viability. Results from this test (Table 3A) shows no toxicity in the tested S. typhimurium strains TA98 and TA100 at concentrations of 5.0 mg/plate, with or without S9 metabolic system (Waleh et al., 1982). Therefore, the highest concentration of freeze-dried E. purpurea flower extract for the mutagenicity/antimutagenicity assay was arbitrarily selected as 5 mg/plate. Mutagenesis occurs spontaneously or may be induced by physical, chemical and biological agents. Research evidences have indicated that free radicals can damage DNA and cause cytotoxicity
and mutagenicity (Zahin, Aqil, & Ahmad, 2010). Echinacea extracts have been used for years and it is reported to be safe for the tested microorganisms (S. typhimurium TA98, TA100, TA1535, TA1537, and TA1538), mammalian cells and mice cells as well as the tested rats and mice (Mengs, Clare, & Poiley, 1991). However, a recent study of Caillet, Lessard, Lamoureux, and Lacroix (2011) indicated that a commercial product of Echinacea (Echinaforce) exhibited mutagenic activity in S. typhimurium strain TA 1535/pSK1002. But they didn’t provide the detailed composition profile for the tested Echinaceas extract product. Nevertheless, their results did raise some concern about the safety of Echinaceas extract and the potential hazards resulting from the long-term use of such plant extract. In the present study, we found that in the presence of the different concentrations of 50% ethanolic E. purpurea flower extracts, the mutation frequencies for the tested S. typhimurium strains TA98 and TA100 did not change significantly when compared to spontaneous mutation frequencies (Table 3B). Indeed, none of the tested concentrations induced a significant increase in the revertant number of TA98 and TA100 strains with or without S9 mix. It appears that the DNA is not a relevant target for 50%
100 Antimutagenic effect (% Inhibition)
increased proportionally with the increases of flower extract and atocopherol from 0 to 20 mg/ml, and with the increases of ascorbic acid and BHA from 0 to 10 mg/ml. At level of 10 mg/ml, the scavenging abilities of samples on DPPH radicals are in the descending order of ascorbic acid > BHA > flower extract > a-tocopherol. In contrast with the 94.40% DPPH scavenging ability generated by the ascorbic acid at 10 mg/ml, a comparable scavenging ability of 95.08% was found for BHA at 20 mg/ml. At level of 30 mg/ml, the flower extract and a-tocopherol showed 90.82 and 93.10% scavenging abilities, respectively. The radical scavenging ability of E. purpurea flower extract could be attributable to the caffeic acid derivatives, especially cichoric acid with two adjacent hydroxyl groups of its phenolic rings, showed the highest radical scavenging ability (Pellati et al., 2004). The order of potency against DPPH radicals was the following: echinacoside > cichoric acid > cynarin > chlorogenic acid > caffeic acid > caftaric acid. The reducing power of flower extract and standards are illustrated in Fig. 1B. The maximum reducing power for ascorbic acid was observed when the concentration was increased to 100 mg/ml. However, the reducing power did not increase proportionally with increasing ascorbic acid concentration. At the level of 100 mg/ml, the reducing power of samples are in the descending order of ascorbic acid > BHA > flower extract > a-tocopherol. The ascorbic acid, BHA, and flower extract attained the same maximum reducing power (2.3 AU) at 100, 200, and 400 mg/ml, respectively. Ferrous ions are the most effective pro-oxidants in the food system, therefore it is frequently used as an antioxidant assessment index (Ebrahimzadeh, Pourmorad, & Bekhradnia, 2008; Yamaguchi, Tatsumi, Kato, & Yoshimitsu, 1988). The ferrous ions chelating ability of flower extracts and standards are illustrated in Fig. 1C. The flower extracts exerted an 81.88% chelating effect at 3 mg/ml concentration when compared with the EDTA concentration of 10 mg/ml. The chelating ability was not enlarged when the concentration was increased to 5 mg/ml, indicating a saturation state was almost attained at the 3 mg/ml of flower extract. However, the ferrous ions chelating ability of ascorbic acid, BHA and a-tocopherol were not detectable. The EDTA was more potent than E. purpurea flower extract on ferrous ions chelating ability. The ferrous ions chelating ability of E. purpurea flower extracts may be attributed to the specific functional groups in its flavanol structure. The adjacent hydroxyl and carbonyl groups in the molecule or hydroxyl groups among molecules could chelate ferrous ion to form a complex (Shahidi & Wanasundara, 1992). Therefore, the more the hydroxyl and carbonyl groups were in appropriate positions, the higher the chelating ability of the molecule could exhibit.
A
80
60 Strong 40 Moderate 20
Weak
0 0
1
2
3
4
5
Concentration of extracts (mg/plate)
B
100 Antimutagenic effect (% Inhibition)
174
80
60 Strong 40 Moderate 20
Weak
0 0
1
2
3
4
5
Concentration of extracts (mg/plate) Fig. 2. Inhibitory effect of ethanolic extract from freeze-dried E. purpurea flower against the mutagenicity of mutagens to Salmonella typhimurium TA98 and TA100. (A) S. typhimurium TA98, -C- direct mutagen, 4-nitro-o-phenylenediamine without S9 mix; -B- indirect mutagen, 2-aminoanthracene with S9 mix. (B) S. typhimurium TA100, -:- direct mutagen, sodium azide without S9 mix; -6- indirect mutagen, 2aminoanthracene with S9 mix. Each value is expressed as mean standard deviation (n ¼ 3).
Y.-L. Tsai et al. / LWT - Food Science and Technology 46 (2012) 169e176
ethanolic E. purpurea flower extracts and they do not produce DNA lesions. Thus, the E. purpurea flower ethanolic extract appears to be non-genotoxic to the tested TA98 and TA100 strains. The Ames test has been widely used to assess the antimutagenic activity of various compounds (Marnewick, Wentzel, Gelderblom, & Joubert, 2000; Zahin et al., 2010). In this study, 2-AA was used as indirect mutagen requiring metabolic activation, and 4-NP and NaN3 were used as direct-acting mutagens. The E. purpurea flower ethanolic extract markedly and dose-dependently decreased the mutagenicity of 2-AA in the Ames test with the S. typhimurium TA98 and TA100 strains (Fig. 2). The freeze-dried E. purpurea flower ethanolic extract in the range of 0.25e5.0 mg/plate showed 30.85e71.90% inhibitory effects on 2-AA toward TA98, and showed 30.57e70.27% inhibitory effect on 2-AA toward TA100 (Fig. 2). On the other hand, no inhibitory effect on 4-NP toward TA98 was noted with freeze-dried E. purpurea flower ethanolic extract (Fig. 2). Nevertheless, the freeze-dried E. purpurea flower ethanolic extract exhibited 0e9.91% inhibitory effect on NaN3 toward TA100. In the case of TA98, no inhibitory effect was shown by extract at 0.25e5.0 mg/plate. In the case of TA100, a weak inhibitory effect was shown by extract at 2.5e5.0 mg/plate, and it showed no inhibitory effects at 0.25e1.0 mg/plate. The freeze-dried E. purpurea flower ethanolic extract tended to inhibit the mutagenicity of indirect-acting mutagen that required metabolic activation of S9 mix more markedly than that of direct-acting mutagen. Some studies have been reported with possible explanation for the mechanism of protection against mutagens (Marnewick et al., 2000; Yen & Chen, 1995). Yen and Chen (1995) indicated that compounds with antioxidant activity could inhibit mutation and cancer due to the fact that they can scavenge a free radical or induce an antioxidant enzyme. Marnewick et al. (2000) reported that tea components could directly interact with the genotoxic reactive intermediates that may result in the prevention of mutagenesis. Furthermore, the flavanol structures in catechins provide a nucleophilic characteristic to react with an electrophilic mutagen to form flavanol-mutagen adducts which may prevent the occurrence of mutagenicity (Stich, 1991). In this study, freeze-dried E. purpurea flower ethanolic extract exhibits good scavenging effect on free radicals, reducing power and metal-binding ability. Thus, it appears that these relevant bioactive components and properties of freezedried E. purpurea flower ethanolic extract may contribute to antimutagenic potencies against different types of chemical mutagen. 4. Conclusion The extraction yield, total phenols, individual and total caffeic acid derivatives extracted from freeze-dried E. purpurea flowers were affected by ethanol/water ratio and extraction temperature. When the extraction yields of bioactive compounds were taken into consideration, the optimal extraction conditions of E. purpurea flowers were 50% ethanol and 65 C of extracting temperatures. The 50% ethanolic extract of E. purpurea flowers were good in antioxidant properties, and did not show toxicity and mutagenicity toward S. typhimurium test strains TA98 and TA100 with the concentrations that enabled antimutagenic activity. Furthermore, E. purpurea flowers ethanolic extract exhibits strong antimutagenicity when TA98 and TA100 were used as experimental microorganisms in the presence of metabolic activator. Acknowledgment The authors would like to thank National Science Council of Taiwan for financially supporting this research under Grant No. NSC-94-2214-E-241-002 and NSC-95-2221-E-241-002.
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References Alothman, M., Bhat, R., & Karim, A. A. (2009). Antioxidant capacity and phenolic content of selected tropical fruits from Malaysia, extracted with different solvents. Food Chemistry, 115, 785e788. Barnes, J., Anderson, L. A., Gibbons, S., & Phillipson, J. D. (2005). Echinacea species (Echinacea angustifolia (DC.) Hell., Echinacea pallida (Nutt.) Nutt., Echinacea purpurea (L.) Moench): a review of their chemistry, pharmacology and clinical properties. Journal of Pharmacy and Pharmacology, 57(8), 929e954. Bauer, R., & Wagner, H. (1991). Echinacea species as potential immunostimulating drugs. In H. Wagner, & N. R. Farnsworth (Eds.), Economic and medicinal plant research, Vol. 5 (pp. 253e321). New York: Academic Press. Caillet, S., Lessard, S., Lamoureux, G., & Lacroix, M. (2011). Umu test applied for screening natural antimutagenic agents. Food Chemistry, 124, 1699e1707. Camel, V. (2000). Microwave-assisted solvent extraction of environmental samples. Trends in Analytical Chemistry, 19, 229e248. Dinis, T. C. P., Madeira, V. M. C., & Almeida, L. M. (1994). Action of phenolic derivatives (acetaminophen, salicylate, and 5-amino salicylate) as inhibitors of membrane lipid peroxidation and as peroxyl radical scavengers. Archives of Biochemistry and Biophysics, 315, 161e169. Ebrahimzadeh, M. A., Pourmorad, F., & Bekhradnia, A. R. (2008). Iron chelating activity, phenol and flavonoid content of some medicinal plants from Iran. African Journal of Biotechnology, 7, 3188e3192. Hu, C., & Kitts, D. D. (2000). Studies on the antioxidant of Echinacea root extract. Journal of Agricultural and Food Chemistry, 48, 1466e1472. Ikken, Y., Morales, P., Martinez, A., Marin, M. L., Haza, A. I., & Cambero, M. I. (1999). Antimutagenic effect of fruit and vegetable ethanolic extracts against N-nitrosamines evaluated by the Ames test. Journal of Agricultural and Food Chemistry, 47, 3257e3264. Kim, S. J., Murthy, H. N., Hahn, E. J., Lee, H. L., & Paek, K. Y. (2007). Parameters affecting the extraction of ginsenosides from the adventitious roots of ginseng (Panax ginseng C.A. Meyer). Separation and Purification Technology, 56, 401e406. Lin, S. D., Sung, J. M., & Chen, C. L. (2011). Effect of drying and storage conditions on caffeic acid derivatives and total phenolics of Echinacea Purpurea grown in Taiwan. Food Chemistry, 125, 226e231. Marnewick, J. L., Wentzel, C. A., Gelderblom, C. A., & Joubert, E. (2000). An investigation on the antimutagenic properties of South African herbal teas. Mutation Research, 471, 157e166. Maron, D. M., & Ames, B. N. (1983). Revised methods for the Salmonella mutagenicity test. Mutation Research, 113, 173e215. Mengs, U., Clare, C. B., & Poiley, J. A. (1991). Toxicity of Echinacea purpurea. Acute, subacute and genotoxicity studies. Arzneimittel-Forschung/Drug Research, 41, 1076e1081. Mortelmans, K., & Zeiger, E. (2000). The Ames Salmonella/microsome mutagenicity assay. Mutation Research, 455, 29e60. Ong, T., Wong, W. Z., Stewart, J. D., & Brockman, H. E. (1986). Chlorophyllin: a potent antimutagen against environmental and dietary complex mixtures. Mutation Research, 173, 111e115. Oyaizu, M. (1986). Studies on products of browning reactions: antioxidative activities of products of browning reaction prepared from glucosamine. Japanese Journal of Nutrition, 44, 307e315. Pellati, F., Benvenuti, S., Magro, L., Melegari, M., & Soragni, F. (2004). Analysis of phenolic compounds and radical scavenging activity of Echinacea spp. Journal of Pharmaceutical and Biomedical Analysis, 35, 289e301. Perry, N. B., Burgess, E. J., & Glennie, V. A. (2001). Echinacea standardization: analytical methods for phenolic compounds and typical levels in medicinal species. Journal of Agriculture and Food Chemistry, 49, 1702e1706. Shahidi, F., & Wanasundara, P. K. J. (1992). Phenolic antioxidants. Critical Reviews in Food Science and Nutrition, 32, 67e103. Shimada, K., Fujikawa, K., Yahara, K., & Nakamura, T. (1992). Antioxidative properties of xanthan on the autoxidation of soybean oil in cyclodextrin emulsion. Journal of Agricultural and Food Chemistry, 40, 945e948. Spelman, K., Wetschler, M. H., & Cech, N. B. (2009). Comparison of alkylamide yield in ethanolic extracts prepared from dry Echinacea purpurea utilizing HPLC-ESIMS. Journal of Pharmaceutical and Biomedical Analysis, 49, 1141e1149. Spigno, G., & De Faveri, D. M. (2007). Antioxidants from grape stalks and marc: influence of extraction procedure on yield, purity and antioxidant power of the extracts. Journal of Food Engineering, 78, 793e801. Stanisavljevi c, I., Stoji cevi c, S., Veli ckovi c, D., Veljkovi c, V., & Lazi c, M. (2009). Antioxidant and antimicrobial activities of Echinaces (Echinacea purpurea L.) extracts obtained by classical and ultrasound extraction. Chinese Journal of Chemical Engineering, 17, 478e483. Stich, H. F. (1991). The beneficial and hazardous effects of simple phenolic compounds. Mutation Research, 259, 307e324. Stuart, D. L., & Wills, R. B. H. (2000). Factors affecting the extraction of alkylamides and cichoric acid during ethanolic processing of Echinacea purpurea (L.) Moench. Australian Journal of Experimental Agriculture, 40, 873e877. Taiwan Food and Drug Administration. (2011). Echinacea purpurea. http://consumer. fda.gov.tw/Food/Material.aspx?nodeID¼160#. Thygesen, L., Thulinn, J., Mortensen, A., Skibsted, L. H., & Molgaard, P. (2007). Antioxidant activity of cichoric acid and alkamides from Echinacea purpurea, alone and in combination. Food Chemistry, 101, 74e81. Waleh, N. S., Rapport, S. J., & Mortelmans, K. E. (1982). Development of a toxicity test to be coupled to the Ames Salmonella assay and the method of construction of the required strains. Mutation Research, 97, 247e256.
176
Y.-L. Tsai et al. / LWT - Food Science and Technology 46 (2012) 169e176
Wang, L., & Weller, C. L. (2006). Recent advances in extraction of nutraceuticals from plants. Trends in Food Science and Technology, 17, 300e312. Wu, C. H., Murthy, H. N., Hahn, E. J., Lee, H. L., & Paek, K. Y. (2008). Efficient extraction of caffeic acid derivatives from adventitious roots of Echinacea purpurea. Czech Journal of Food Sciences, 26, 254e258. Xi, J., Shen, D., Zhao, S., Lu, B., Li, Y., & Zhang, R. (2009). Characterization of polyphenols from green tea leaves using a high hydrostatic pressure extraction. International Journal of Pharmaceutics, 382, 139e143. Yam, T. S., Shah, S., & Hamilton-Miller, J. M. T. (1997). Microbiology activity of whole and fractionated crude extracts of tea (Camellia sinensis), and of tea components. FEMS Microbiology Letters, 152, 169e174.
Yamaguchi, R., Tatsumi, M. A., Kato, K., & Yoshimitsu, U. (1988). Effect of metal salts and fructose on the autoxidation of methyl linoleate in emulsions. Agricultural and Biological Chemistry, 52, 849e850. Yen, G. C., & Chen, H. Y. (1995). Antioxidant activity of various tea extracts in relation to their antimutagenicity. Journal of Agricultural and Food Chemistry, 43, 27e32. Zahin, M., Aqil, F., & Ahmad, I. (2010). Broad spectrum antimutagenic activity of antioxidant active fraction of Punica granatum L. peel extracts. Mutation Research, 703, 99e107. Zhai, Z., Haney, D., Wu, L., Solco, A., Murphy, P. A., Wurtele, E. S., et al. (2007). Alcohol extracts of Echinacea inhibit production of nitric oxide and tumor necrosis factoralpha by macrophages in vitro. Food Agriculture Immunology, 18, 221e236.
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LWT - Food Science and Technology journal homepage: www.elsevier.com/locate/lwt
Caffeic acid derivatives, total phenols, antioxidant and antimutagenic activities of Echinacea purpurea flower extracts Yu-Ling Tsai a, b, Shiow-Ying Chiou c, Kung-Chi Chan b, Jih-Min Sung c, Sheng-Dun Lin c, * a
Department of Nutrition, Hungkuang University, 34 Chung-Chie Road, Shalu, Taichung 43302, Taiwan, ROC Department of Food and Nutrition, Providence University, 200 Chung-Chie Road, Shalu, Taichung 43301, Taiwan, ROC c Department of Food Science and Technology, Hungkuang University, 34 Chung-Chie Road, Shalu, Taichung 43302, Taiwan, ROC b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 May 2011 Received in revised form 27 July 2011 Accepted 30 September 2011
This study evaluated the effects of ethanol concentration and extraction temperature on the quality of extracts obtained from freeze-dried Echinacea purpurea flowers. The antioxidant and antimutagenic effects of flower extract were also examined. The optimal extraction conditions for freeze-dried flowers were 50% aqueous ethanol and 65 C extraction temperature, with extraction yield of 37.4%. The contents of total phenols, caffeic acid derivatives, and cichoric acid in freeze-dried extracts were 473.34 mg chlorogenic acid equivalents/g, 302.20 mg/g and 217.61 mg/g, respectively. The 50% ethanolic flower extract did not show toxicity and mutagenicity toward Salmonella typhimurium TA98 and TA100 with or without S9 mix. The ethanolic extract at 0.25e5 mg/plate exhibits a dose-dependent inhibitory effect against the mutagenicity of 2-aminoanthracene. Thus, freeze-dried E. purpurea flower ethanolic extract exhibits good antioxidant and antimutaginic activities. Ó 2011 Elsevier Ltd. All rights reserved.
Keywords: Echinacea purpurea Caffeic acid derivative Antioxidant Antimutagenicity
1. Introduction Echinacea purpurea L. is one of the top selling medicinal plants widely used to alleviate colds, sore throats and other upper respiratory infections. The prepared Echinacea products (e.g., infusions, tinctures and capsules) are used to stimulate immune system, and their immuno-stimulating properties are attributed to the bioactive phytochemicals including caffeic acid derivatives, alkamides, polysaccharides, and glycoproteins. Among these phytochemicals, caffeic acid derivatives, especially cichoric acid, possess many bioactive functions including anti-hyaluronidase activity, protection of collagen from free radical induced degradation, antiviral activity, inhibition of human immunodeficiency virus type 1 integrase and replication, promoting phagocyte activity in vitro and in vivo and a high free radical scavenging property (Barnes, Anderson, Gibbons, & Phillipson, 2005; Bauer, & Wagner, 1991; Pellati, Benvenuti, Magro, Melegari, & Soragni, 2004). Thus, cichoric acid is commonly used as marker to determine the medicinal quality of Echinacea products (Thygesen, Thulinn, Mortensen, Skibsted, & Molgaard, 2007). Various extraction techniques have been developed to obtain phytochemicals from plant materials (Wang & Weller, 2006). Both
* Corresponding author. Tel.: þ886 4 2631 8652x5038; fax: þ886 4 2631 9176. E-mail address: [email protected] (S.-D. Lin). 0023-6438/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.lwt.2011.09.026
the classical extraction methods including solvent extraction with or without mechanical agitation (or shaking) and some novel techniques such as ultrasound-assisted extraction and microwaveassisted extraction are fast and efficient for extracting phytochemicals from plant materials. The ultrasound-assisted extraction and microwave-assisted extraction have many advantages over classical extraction methods in terms of extraction time, solvent consumption, extraction yields and reproducibility. However, these novel techniques have only been tested in a limited field of applications (Wang & Weller, 2006). Nevertheless, from industrial-scale production viewpoint, the selected extraction technique must be versatile, simple, low-cost and safe for both the operating personnel and the consumers (Wu, Murthy, Hahn, Lee, & Paek, 2008). In Taiwan, the classical solvent extraction coupled with higher temperature has been commercially used to obtain bioactive compounds from various plant species for years. Type of solvent is an important parameter affecting the recovery of phytochemicals during extractions (Wang & Weller, 2006). Alcoholic solvents, such as ethanol and methanol at different concentrations in water, have been used to extract phenols from plant materials (Alothman, Bhat, & Karim, 2009). Pellati et al. (2004) found that methanol/water extraction was more efficient than ethanol/water extraction for extracting total phenols in Echinacea. However, ethanol is non-toxic, it is easy to be recycled and mixed with water in different ratios. Besides, the acceptability of ethanol and water for human consumption models is high.
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Extraction temperature is another important parameter needs to be optimized during extraction (Camel, 2000; Kim, Murthy, Hahn, Lee, & Paek, 2007; Stuart & Wills, 2000; Wang & Weller, 2006). An increase in the working temperature generally favors extraction on both the solubility of solute and the diffusion coefficient, but also that beyond a certain value phenolic compounds can be denatured (Spigno, & De Faveri, 2007). E. purpurea has recently been introduced into Taiwan and grows well, and its flower was recently permitted for use as a food material by the Taiwan government (Taiwan Food and Drug Administration, 2011). The total phenols and caffeic acid derivatives contents in different plant parts were in the descending order: flowers > leaves > stems > roots (Lin, Sung, & Chen, 2011). However, little information is available regarding the phytochemicals, antioxidant and antimutagenic activities of E. purpurea flower extracts. Therefore, the objectives of this study were to determine the effects of various concentrations of ethanol and extraction temperatures on the extraction yields, total phenols, and caffeic acid derivatives of freeze-dried E. purpurea flowers, and to investigate the antioxidant properties of the ethanolic extract of E. purpurea flowers. The antimutagenic effects of E. purpurea flower extract by using Salmonella typhimurium test (Ames test) were also determined. 2. Materials and methods 2.1. Materials The flowers of E. purpurea (L.) Moench variety CLS-P2 harvested from six-month-old plants were donated by Echili Biotechnology (Taichung, Taiwan). The flowers were freeze-dried with vacuum on the same day as harvested. The dried flowers were ground in a mill, and screened through a 0.5 mm sieve. The dried flower powders were stored in sealed PET/Al/PE bag for 9 months at 20 C. Methanol, acetonitrile and phosphoric acid were purchased from Mallinckrodt Baker, Inc. (New Jersey, USA). Caftaric acid, echinacoside, cichoric acid and cynarin were purchased from ChromaDex Inc. (Santa Ana, CA, USA). Chlorogenic acid, sodium tungstate dehydrate, phosphomolybdic acid hydrate 1,1-diphenyl2-picrylhydrazyl (DPPH), trichloroacetic acid, potassium ferricyanide, ferrous chloride, ferrozine, ascorbic acid, a-tocopherol, butylated hydroxyanisole (BHA), ethylenediaminetetraacetic acid (EDTA), 2-aminoanthracene (2-AA), biotin, dimethyl sulfoxide (DMSO), glucose-6-phosphate (G-6-P), histidine, 4-nitro-o-phenylenediamine (4-NP) and sodium azide (NaN3) were purchased from SigmaeAldrich Chemical Co. (St. Louis, MO, USA). Anhydrous sodium carbonate was purchased from Shimakyu’s Pure Chemicals (Osaka, Japan). Sodium phosphate and ferric chloride were purchased from Wako Pure Chemical Industries Co. (Osaka, Japan). A metabolic activation fraction (S9 mix) prepared from SpragueeDawley rat liver was purchased from Nature Opera Biotechnology Inc. (Taipei, Taiwan). Ethanol (95%) was purchased from Taiwan Tobacco & Liquor Co. (Tainan, Taiwan). 2.2. Ethanol extracts preparation 2.2.1. Effect of various concentrations of ethanol The reverse osmosis (RO) water was prepared from tap water by RO machine system (Lepa Industrial Co., Ltd., Taipei, Taiwan). The 25%, 50%, and 75% aqueous ethanol were prepared from 95% ethanol mixed with RO water. The ethanolic extracts of E. purpurea flowers were obtained by extracting the ground flower powder (10 g) with 100 ml of RO water, 25%, 50%, 75%, and 95% aqueous ethanol in a shaking bath (25 C) (SB302, Kansin Instruments Co., Kaohsiung, Taiwan) at 100 rpm for 30 min, and then centrifuged at
3460g for 10 min, filtered through Whatman No.1 filter paper. The residue was re-extracted with two extra 100 ml portions of solvent as described above. The combined ethanolic extracts were rotary evaporated at 40 C and then freeze-dried with vacuum. Dry extracts thus obtained were stored at 20 C before use. The extracts extracted with 0%, 25%, 50%, 75%, and 95% ethanol were designated as E0, E25, E50, E75, and E95, respectively. All treatments were randomly produced. All experiments were done in triplicate. 2.2.2. Effect of various extraction temperatures The flower powders (10 g for each sample) were extracted with 100 ml of 50% aqueous ethanol in a shaking bath at 100 rpm for 30 min under 25 C, 35 C, 45 C, 55 C, 65 C, 75 C, 85 C, or 95 C conditions, and then centrifuged at 3460 g for 10 min, filtered through Whatman No.1 filter paper. The residue was re-extracted with two extra 100 ml portions of solvent as described above. The combined ethanolic extracts were rotary evaporated at 40 C and then freeze-dried with vacuum. Dry extracts thus obtained were stored at 20 C before use. The extracts prepared under 25 C, 35 C, 45 C, 55 C, 65 C, 75 C, 85 C, or 95 C conditions were designated as ET25, ET35, ET45, ET55, ET65, ET75, ET85, and ET95, respectively. All experiments were done in triplicate. 2.3. Determination of total phenols The content of total phenols in the extracts was determined using the Folin-Denis method (Perry, Burgess, & Glennie, 2001) with minor modifications. Folin-Denis reagent was prepared according to the method of Perry et al. (2001). Each extract (100 mg) was dissolved in 70% ethanol (4.0 ml) using an ultrasonic bath with 40 kHz for 1 min, and then the volume was adjusted to 5 ml. FolineDenis reagent (2.5 ml) was added to either 0.25 ml of extract or standard solution. After 3 min 35% sodium carbonate solution (2.5 ml) was added and the test solution was made up to 25 ml with H2O and mixed. After 45 min of incubation in room temperature, the test solution was centrifuged at 3460 g for 10 min. The absorption value was determined at 745 nm, with chlorogenic acid used as a standard. The total phenols were expressed as mg chlorogenic acid equivalents per g dry extract [the equation of standard curve: absorbance at 745 nm ¼ 0.5441 Cchlorogenic acid (mg/ ml) þ 0.0441, R2 ¼ 0.9963]. Each analysis was carried out in triplicate. 2.4. Determination of caffeic acid derivatives The content of caffeic acid derivatives in the extracts was determined according to the methods of Hu and Kitts (2000) and Pellati et al. (2004) with minor modifications. Briefly, each extract (100 mg) was dissolved in 70% ethanol (4.0 ml) using an ultrasonic bath with 40 kHz for 1 min, and then the volume was adjusted to 5 ml. The solution was filtered using a PVDF syringe filter (13 mm 0.45 mm) prior to injection onto an HPLC. The HPLC system consisted of a Hitachi L-2130 pump, a Hitachi L-2400 UV detector, and a LiChrospherÒ 100 RP-18e column (250 mm 4.6 mm 5 mm, Merck Co., Germany). The mobile phase was (A) acetonitrile/water containing 0.1% H3PO4 (10:90) and (B) acetonitrile/water containing 0.1% H3PO4 (25:75). A gradient elution profile was used with B increasing from 0% to 100% in 30 min and maintained at 100% for 10 min. Then a linear gradient of 100% B decreased to 0% B in 10 min. The flow rate was 1.5 ml/min, and the wavelength of the UVevisible detector was set at 330 nm. The sample injection volume was 20 ml. Contents of various caffeic acid derivatives were calculated on the basis of the calibration
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Table 1 Extraction of freeze-dried E. purpurea flowers with various concentrations of ethanol at 25 C: Extraction yield, content of total phenolics and caffeic acid derivatives in dry extracts. Extraction method
Extraction yield (%)a
E0 E25 E50 E75 E95
39.8 36.9 34.3 28.0 3.3
1.3Ab 0.9B 0.1C 1.2D 0.6E
Content (mg/g extract) Caftaric acid
Chlorogenic acid
Echinacoside
16.56 42.71 67.07 50.04 16.35
0.14 0.68 2.41 2.84 0.53
0.53 0.79 1.02 1.07 0.54
0.23D 3.86C 0.67A 1.39B 0.27D
0.01C 0.15B 0.01A 0.10A 0.02B
0.06C 0.03B 0.09A 0.07A 0.04C
Cichoric acid 37.23 108.86 180.29 160.95 42.54
0.25D 4.61C 1.76A 5.12B 0.36D
Cynarin
Total caffeic acid derivatives
nd nd nd nd nd
54.46 153.04 250.79 214.90 59.96
0.19D 4.33C 2.42A 5.12B 0.41D
Total phenolics 97.07 282.78 441.33 399.82 104.47
6.91D 6.44C 3.54A 8.37B 3.80D
Means with different capital letters in the same column are significantly different (P < 0.05). nd: not detected. a Extraction yield (%) ¼ (dried extract weight/sample weight) 100%. b Each value is expressed as mean SD (n ¼ 3).
curve of each caftaric acid, chlorogenic acid, echinacoside, cichoric acid and cynarin. Each analysis was carried out in triplicate. 2.5. Antioxidant properties 2.5.1. Scavenging ability on 1,1-diphenyl-2-picrylhydrazyl radicals For DPPH radicals scavenging ability assay (Shimada, Fujikawa, Yahara, & Nakamura, 1992), each extract (10e50 mg/ml, 4 ml) in methanol was mixed with 1 ml of methanolic solution containing DPPH radicals, resulting in a final concentration of 0.2 mM DPPH. The mixture was shaken vigorously and left to stand for 30 min in the dark, and the absorbance of the mixture was then measured at 517 nm against a blank. Ascorbic acid, BHA, and a-tocopherol were used for comparison. 2.5.2. Reducing power The reducing power was determined according to the method of Oyaizu (1986). Each extract (100e500 mg/ml, 2.5 ml) in methanol was mixed with 2.5 ml of 200 mM sodium phosphate buffer (pH 6.6) and 2.5 ml of 1% potassium ferricyanide, and the mixture was incubated at 50 C for 20 min. After 2.5 ml of 10% trichloroacetic acid (w/v) were added, the mixture was centrifuged at 2600 g for 10 min. The upper layer (5 ml) was mixed with 5 ml of deionized water and 1 ml of 0.1% ferric chloride, and the absorbance of the mixture was measured at 700 nm against a blank to determine the amount of ferric ferrocyanide (Prussian blue) formed. Ascorbic acid, BHA, and a-tocopherol were used for comparison. 2.5.3. Ferrous ions chelating abilities Chelating ability was determined according to the method of Dinis, Madeira, and Almeida (1994). Each extract (1e5 mg/ml, 1 ml)
in methanol was mixed with 3.7 ml of methanol and 0.1 ml of 2 mM ferrous chloride. After 30 s of standing, 0.2 ml of 5 mM ferrozine was added. After 10 min at room temperature, the absorbance of the mixture was determined at 562 nm against a blank. The EDTA, ascorbic acid, BHA, and a-tocopherol were used for comparison. 2.6. Ames test 2.6.1. Salmonella strains The histidine-requiring strains of S. typhimurium TA 98 and TA 100 were purchased from the Bioresources Collection and Research Center (BCRC), Food Industry Research and Development Institute (FIRDI, Hsinchu, Taiwan). The test strains were characterized for spontaneous reversion and genetic analysis including histidine requirement (his-), UV (uvrB) sensitivity, crystal violet (rfa) sensitivity and ampicillin resistance (Mortelmans & Zeiger, 2000). 2.6.2. Toxicity test For toxicity measurement (Waleh, Rapport, & Mortelmans, 1982), the 50% ethanolic extracts (0.1 ml) from freeze-dried E. purpurea flower (0e5 mg/plate), 0.5 ml of S9 mix or phosphate buffer saline (PBS, 0.1 M, pH 7.4) were mixed with 0.1 ml PBS and 0.1 ml of 12 h-cultured test strain (approximately 108 cells/ml). After incubation for 20 min at 37 C, the serial dilutions were made with nutrient broth no. 2 (OXOID), an aliquot (1 ml) was mixed with 15 ml of nutrient agar and incubated at 37 C for 48 h, and the number of colonies was counted. 2.6.3. Mutagenicity test Mutagenicity was assayed by the standard Ames test (standard plate incorporation assay) (Maron & Ames, 1983; Mortelmans &
Table 2 Extraction of freeze-dried E. purpurea flowers with 50% ethanol at various extraction temperatures: Extraction yield, content of total phenolics and caffeic acid derivatives in dry extracts. Extraction method
Extraction yield (%)a
ET25 ET35 ET45 ET55 ET65 ET75 ET85 ET95
34.3 34.4 35.7 36.4 37.4 38.2 39.0 39.8
0.1Gb 0.4G 0.5F 0.2E 0.6D 0.4C 0.3B 0.3A
Content (mg/g extract) Caftaric acid 67.07 67.96 72.13 72.79 78.18 75.59 74.06 72.43
0.67D 0.99D 1.83C 0.60BC 1.39A 1.11AB 1.42BC 0.66C
Chlorogenic acid 2.41 2.37 3.36 3.84 4.27 3.99 3.42 2.79
0.01E 0.03E 0.31C 0.07B 0.04A 0.12B 0.16C 0.09D
Echinacoside 1.02 1.53 2.16 2.03 2.14 1.98 1.83 1.65
0.09E 0.15D 0.20A 0.00A 0.05A 0.00AB 0.02BC 0.03CD
Means with different capital letters in the same column are significantly different (P < 0.05). nd: not detected. a Extraction yield (%) ¼ (dried extract weight/sample weight) 100%. b Each value is expressed as mean SD (n ¼ 3).
Cichoric acid 180.29 182.98 191.22 191.86 217.61 205.99 196.49 186.35
1.76E 3.95E 1.86CD 1.19CD 4.50A 2.20B 2.54C 4.39DE
Cynarin
Total caffeic acid derivatives
Total phenolics
nd nd nd nd nd nd nd nd
250.79 2.42E 254.84 3.07E 268.87 3.58CD 270.52 1.72CD 302.20 5.98A 287.55 0.97B 275.80 3.83C 263.22 5.17D
441.33 463.00 464.50 466.92 473.34 458.02 456.16 434.41
3.54E 3.54BCD 3.30BC 2.71AB 2.42A 1.73CD 3.77D 3.08E
Y.-L. Tsai et al. / LWT - Food Science and Technology 46 (2012) 169e176
2.6.4. Antimutagenicity assay For the antimutagenicity test, the 50% ethanolic extract from freeze-dried E. purpurea flower and one of three mutagens for each strain were assayed. The positive controls using 4-NP (1.25 mg/ plate) for TA98 strain and NaN3 (1.25 mg/plate) for TA100 strain were tested without S9 metabolic activation whereas those using 2-AA for both TA98 (0.25 mg/plate) and TA 100 (2 mg/plate) strains was tested with S9 activation. The mutagen in PBS (direct test) or S9 mix (indirect test) was added to bacterial culture with the ethanolic extract. After incubation for 20 min at 37 C, the mixture was added to 2.0 ml of top agar and plated as described above. Positive and negative controls were also conducted in each assay. The mutagenicity of appropriate mutagen for each strain in the absence of test samples was defined as 0% inhibition. The % inhibition was calculated as follows according to the method of Ong, Wong, Stewart, and Brockman (1986): % inhibition ¼ [1 (T S)/(M S)] 100, where T is the number of revertants per plate in the presence of mutagen and the test sample, M is the number of revertants per plate in the positive control and S is the number of spontaneous revertants per plate. The inhibition of 25e40% was considered as moderate antimutagenic effect and that of more than 40% was the strong effect. However, the inhibition of less than 25% were considered as weak and was not recognized as a positive result (Ikken et al., 1999).
3. Results and discussion 3.1. Effect of various concentrations of ethanol on the extraction yields, total phenols and caffeic acid derivatives The extraction yield is a measure of the solvent efficiency to extract specific components from the original materials. In the case of E. purpurea, it will give an idea about the extractability of total phenolics including the caffeic acid derivatives under different extraction conditions. Stanisavljevi c, Stojicevi c, Veli ckovi c, Veljkovi c, and Lazi c (2009) used both classical solvent extraction and ultrasound-assisted extraction to investigate the extraction yield of total phenols in aerial part of E. purpurea L., and found that the extract obtained by classical solvent extraction contained more phytochemicals and had greater antioxidant activities than the
80
60
40
20
0 0
10
20
30
40
50
400
500
4
5
Concentration (µ µg/ml)
B
2.5 2.0 1.5 1.0 0.5 0.0 0
100
200
300
Concentration (µ µg/ml)
100
2.7. Statistical analysis
C
80 Chelating ability (% )
All measurements were carried out in triplicate. All data were subjected to analysis of variance using the Statistical Analysis System software package (SAS Institute, Cary, NC, USA). When a significant difference was found among treatments, Duncan’s multiple range tests were performed to determine the differences among the mean values at the level of a ¼ 0.05.
A
100
Scavenging ability (% )
Zeiger, 2000). Both TA98 and TA100 strains were aerobically grown at 37 C in nutrient broth no. 2 (OXOID) supplemented with ampicillin. An aliquot (0.1 ml) of the overnight incubated bacterial culture was mixed with ethanolic extracts (0.25e5 mg/plate) and 0.5 ml PBS (0.1 M, pH 7.4). After incubation for 20 min at 37 C, the mixture was added to 2.0 ml of top agar containing 0.05 mM biotinhistidine and plated onto glucose minimal agar plates (Mortelmans & Zeiger, 2000). For indirect test, 0.5 ml of S9 mix was supplemented instead of phosphate buffer. The activation system (S9 mix) contained 4% of Aroclor 1254-induced rat liver homogenate S9. The reversion rate was compared to that of control plates treated with 50% DMSO. Hisþ revertants were counted after incubation of the plates at 37 C for 48 h. Subsequently, the number of histidineindependent revertant colonies was numerated. Each assay was conducted in triplicate.
Absorbance at 700 nm
172
60
40
20
0 0
1
2
3
Concentration (mg/ml) Fig. 1. Antioxidant properties of E. purpurea flower extracts, ascorbic acid, butylated hydroxyanisole (BHA), a-tocopherol and ethylenediaminetetraacetic acid (EDTA). (A) scavenging ability on 1,1-dipheny1-2-picrylhydrazyl radicals. (B) reducing power. (C) ferrous ions chelating ability. Flower extract: The extract was prepared from vacuumdried E. purpurea flowers with 50% aqueous ethanol and 65 C extracting temperatures. Each value is expressed as mean standard deviation (n ¼ 3). -C-, E. purpurea extract; -7-, ascorbic acid; ---, a-tocopherol; -A-, BHA; -B-, ethylenediaminetetraacetic acid.
Y.-L. Tsai et al. / LWT - Food Science and Technology 46 (2012) 169e176
173
E. purpurea flowers (Table 1). Therefore, only 50% ethanol was chosen for optimizing extraction temperature.
extract obtained by ultrasonic extraction. They concluded that the results were due to the degradation of total phenols by interaction with the highly reactive hydroxyl radicals formed during sonication. Therefore, in the present study, only the traditional solvent extraction was used to extract the phytochemicals in freeze-dried E. purpurea flowers. Table 1 shows that the extraction yields of freeze-dried E. purpurea flowers were significantly affected by the ethanol concentrations (P < 0.05), with water extraction giving the highest extraction yield. When the ethanol volume percentage in the solvent was increased up to 95%, the extraction yields were decreased from 39.8% to 3.3%. These results are related to solvent polarity and the solubility of components in tested sample (Xi et al., 2009). The contents of total phenols, individual and total caffeic acid derivatives in extracts were also affected by the ethanol concentrations (Table 1). A protocol of 70% ethanol extraction procedure has been proposed for the manufacture of E. purpurea ethanolic extracts (Spelman, Wetschler, & Cech, 2009). In this study, when the ethanol volume percentage was lower than 50%, the contents of total phenols, caftaric acid, chlorogenic acid, echinacoside, cichoric acid, and total caffeic acid derivatives were increased with the increases of ethanol concentrations. However, the total phenols, individual and total caffeic acid derivatives contents in extracts were decreasing when the ethanol concentration was higher than 50%. When the extraction yields were taken into consideration in this study, the total phenols, individual and total caffeic acid derivatives contents (mg/g freeze-dried flowers) of freeze-dried E. purpurea flowers are in the descending order of E50 > E75 y E25 > E0 > E95. As for the caffeic acid derivatives detectable in the freeze-dried flowers extracts (Table 1), cichoric acid (ranged from 68.36 to 74.90%) was the major content, followed by caftaric acid (ranged from 23.09 to 30.41%). This result is in agreement with the report of Zhai et al. (2007). Several studies (Stuart & Wills, 2000; Wu et al., 2008) have shown that cichoric acid and other active ingredients in E. purpurea root are optimally extracted with 60% ethanol. Similarly our results indicate that 50% ethanol is favorable for extracting individual caffeic acid derivatives in freeze-dried
3.2. Effect of various extraction temperatures on the extraction yields, total phenols and caffeic acid derivatives Table 2 shows that extraction yields of freeze-dried E. purpurea flowers ranged from 34.3% at 25 C to 39.8% at 95 C, with the highest extraction yield obtained from the ET95. The contents of total phenols, individual and total caffeic acid derivatives of 50% ethanol extracts were also greatly affected by the extraction temperatures. The total phenols, individual and total caffeic acid derivatives contents of extracts was enhanced by elevated extraction temperatures ranged from 25 C to 65 C. These increments in the active ingredients with the increase of temperature might be the result of an increased diffusivity of the solvent into the cells and an enhanced de-sorption of the components forming the cells (Camel, 2000; Wu et al., 2008). When the extraction yields were taken into consideration, the active ingredients contents of freezedried E. purpurea flowers also has noticeable increase with increasing extraction temperatures of 25e65 C. But the active ingredients contents of extracts showed a reverse trend between 65 C and 95 C of extraction temperatures. When the extraction yields were taken into consideration, total phenols and caftaric acid contents (mg/g freeze-dried flowers) change of flowers were not significantly (P > 0.05), total caffeic acid derivatives, cichoric acid, chlorogenic acid, and echinacoside contents in flowers showed a slightly decrease between 65 C and 95 C of extraction temperatures. In this study, the 65 C of extraction temperature gave higher total phenols, individual and total caffeic acid derivatives than the other extraction temperatures tested. Wu et al. (2008) reported that the treatment of the samples with 60% ethanol at 60 C was proven to be the most suitable procedure. 3.3. Antioxidant properties The DPPH radical scavenging ability of flower extract and standards are reported in Fig. 1A. The DPPH scavenging ability
Table 3 Toxicity and mutagenicity of ethanolic extract from freeze-dried E. purpurea flower toward Salmonella typhimurium TA98 and TA100 with and without S9 mix. Extract (mg/plate)
Survival (%)a TA98
TA100
S9
þS9
S9
(A) Toxicity 0.0 0.25 0.5 1.0 2.5 5.0
100.00Ab 86.19 5.47A 91.32 10.41A 86.44 6.74A 95.11 12.13A 96.72 6.57A
100.00B 97.28 107.76 106.21 115.62 97.77
Extract (mg/plate)
Hisþ revertants/plate (Mutagenicity ratio)c
7.02B 13.25AB 6.99AB 8.18A 4.28B
TA98
33 32 32 34 34 31
100.00B 99.37 108.20 105.47 99.40 91.80
3.51B 5.79A 5.95AB 2.64B 2.28C
100.00C 117.99 116.01 113.95 111.67 107.93
10.57A 0.46AB 5.05AB 1.01AB 1.88BC
TA100
S9 (B) Mutagenicity 0.0 0.25 0.5 1.0 2.5 5.0
þS9
þS9 3 3 4 6 4 7
(1.00)Ab (0.96)A (0.98)A (1.04)A (1.02)A (0.94)A
60 67 59 62 61 55
S9 7 (1.00)A 11 (1.12)A 4 (0.99)A 4 (1.04)A 4 (1.02)A 5 (0.93)A
Means with different capital letter within a column are significantly different (P < 0.05). a Survival (%) ¼ (number of colonies of extract per plate/number of colonies of control per plate) 100%. b Each value is expressed as mean standard deviation (n ¼ 3). c Mutagenicity ratio ¼ induced revertants per plate/spontaneous revertants (control) per plate.
142 140 147 135 141 156
þS9
7 (1.00)A 9 (0.99)A 7 (1.04)A 17 (0.95)A 19 (0.99)A 4 (1.10)A
125 104 113 129 120 125
5 (1.00)A 7 (0.83)A 10 (0.91)A 13 (1.03)A 1 (0.96)A 16 (1.00)A
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3.4. Toxicity, mutagenicity and antimutagenicity Many phenolic compounds were found to possess antimicrobial activity (Yam, Shah, & Hamilton-Miller, 1997). In this study, a good portion of total phenols and caffeic acid derivatives were present in the 50% ethanolic extracts of freeze-dried E. purpurea flower (Table 2). Therefore, a toxic concentration test preceding the antimutagenicity studies was performed. The result shows that the different concentrations (0.25, 0.5, 1.0, 2.5 and 5.0 mg/plate) of freeze-dried E. purpurea flower extract added to Ames indicator bacteria did not influence their viability. Results from this test (Table 3A) shows no toxicity in the tested S. typhimurium strains TA98 and TA100 at concentrations of 5.0 mg/plate, with or without S9 metabolic system (Waleh et al., 1982). Therefore, the highest concentration of freeze-dried E. purpurea flower extract for the mutagenicity/antimutagenicity assay was arbitrarily selected as 5 mg/plate. Mutagenesis occurs spontaneously or may be induced by physical, chemical and biological agents. Research evidences have indicated that free radicals can damage DNA and cause cytotoxicity
and mutagenicity (Zahin, Aqil, & Ahmad, 2010). Echinacea extracts have been used for years and it is reported to be safe for the tested microorganisms (S. typhimurium TA98, TA100, TA1535, TA1537, and TA1538), mammalian cells and mice cells as well as the tested rats and mice (Mengs, Clare, & Poiley, 1991). However, a recent study of Caillet, Lessard, Lamoureux, and Lacroix (2011) indicated that a commercial product of Echinacea (Echinaforce) exhibited mutagenic activity in S. typhimurium strain TA 1535/pSK1002. But they didn’t provide the detailed composition profile for the tested Echinaceas extract product. Nevertheless, their results did raise some concern about the safety of Echinaceas extract and the potential hazards resulting from the long-term use of such plant extract. In the present study, we found that in the presence of the different concentrations of 50% ethanolic E. purpurea flower extracts, the mutation frequencies for the tested S. typhimurium strains TA98 and TA100 did not change significantly when compared to spontaneous mutation frequencies (Table 3B). Indeed, none of the tested concentrations induced a significant increase in the revertant number of TA98 and TA100 strains with or without S9 mix. It appears that the DNA is not a relevant target for 50%
100 Antimutagenic effect (% Inhibition)
increased proportionally with the increases of flower extract and atocopherol from 0 to 20 mg/ml, and with the increases of ascorbic acid and BHA from 0 to 10 mg/ml. At level of 10 mg/ml, the scavenging abilities of samples on DPPH radicals are in the descending order of ascorbic acid > BHA > flower extract > a-tocopherol. In contrast with the 94.40% DPPH scavenging ability generated by the ascorbic acid at 10 mg/ml, a comparable scavenging ability of 95.08% was found for BHA at 20 mg/ml. At level of 30 mg/ml, the flower extract and a-tocopherol showed 90.82 and 93.10% scavenging abilities, respectively. The radical scavenging ability of E. purpurea flower extract could be attributable to the caffeic acid derivatives, especially cichoric acid with two adjacent hydroxyl groups of its phenolic rings, showed the highest radical scavenging ability (Pellati et al., 2004). The order of potency against DPPH radicals was the following: echinacoside > cichoric acid > cynarin > chlorogenic acid > caffeic acid > caftaric acid. The reducing power of flower extract and standards are illustrated in Fig. 1B. The maximum reducing power for ascorbic acid was observed when the concentration was increased to 100 mg/ml. However, the reducing power did not increase proportionally with increasing ascorbic acid concentration. At the level of 100 mg/ml, the reducing power of samples are in the descending order of ascorbic acid > BHA > flower extract > a-tocopherol. The ascorbic acid, BHA, and flower extract attained the same maximum reducing power (2.3 AU) at 100, 200, and 400 mg/ml, respectively. Ferrous ions are the most effective pro-oxidants in the food system, therefore it is frequently used as an antioxidant assessment index (Ebrahimzadeh, Pourmorad, & Bekhradnia, 2008; Yamaguchi, Tatsumi, Kato, & Yoshimitsu, 1988). The ferrous ions chelating ability of flower extracts and standards are illustrated in Fig. 1C. The flower extracts exerted an 81.88% chelating effect at 3 mg/ml concentration when compared with the EDTA concentration of 10 mg/ml. The chelating ability was not enlarged when the concentration was increased to 5 mg/ml, indicating a saturation state was almost attained at the 3 mg/ml of flower extract. However, the ferrous ions chelating ability of ascorbic acid, BHA and a-tocopherol were not detectable. The EDTA was more potent than E. purpurea flower extract on ferrous ions chelating ability. The ferrous ions chelating ability of E. purpurea flower extracts may be attributed to the specific functional groups in its flavanol structure. The adjacent hydroxyl and carbonyl groups in the molecule or hydroxyl groups among molecules could chelate ferrous ion to form a complex (Shahidi & Wanasundara, 1992). Therefore, the more the hydroxyl and carbonyl groups were in appropriate positions, the higher the chelating ability of the molecule could exhibit.
A
80
60 Strong 40 Moderate 20
Weak
0 0
1
2
3
4
5
Concentration of extracts (mg/plate)
B
100 Antimutagenic effect (% Inhibition)
174
80
60 Strong 40 Moderate 20
Weak
0 0
1
2
3
4
5
Concentration of extracts (mg/plate) Fig. 2. Inhibitory effect of ethanolic extract from freeze-dried E. purpurea flower against the mutagenicity of mutagens to Salmonella typhimurium TA98 and TA100. (A) S. typhimurium TA98, -C- direct mutagen, 4-nitro-o-phenylenediamine without S9 mix; -B- indirect mutagen, 2-aminoanthracene with S9 mix. (B) S. typhimurium TA100, -:- direct mutagen, sodium azide without S9 mix; -6- indirect mutagen, 2aminoanthracene with S9 mix. Each value is expressed as mean standard deviation (n ¼ 3).
Y.-L. Tsai et al. / LWT - Food Science and Technology 46 (2012) 169e176
ethanolic E. purpurea flower extracts and they do not produce DNA lesions. Thus, the E. purpurea flower ethanolic extract appears to be non-genotoxic to the tested TA98 and TA100 strains. The Ames test has been widely used to assess the antimutagenic activity of various compounds (Marnewick, Wentzel, Gelderblom, & Joubert, 2000; Zahin et al., 2010). In this study, 2-AA was used as indirect mutagen requiring metabolic activation, and 4-NP and NaN3 were used as direct-acting mutagens. The E. purpurea flower ethanolic extract markedly and dose-dependently decreased the mutagenicity of 2-AA in the Ames test with the S. typhimurium TA98 and TA100 strains (Fig. 2). The freeze-dried E. purpurea flower ethanolic extract in the range of 0.25e5.0 mg/plate showed 30.85e71.90% inhibitory effects on 2-AA toward TA98, and showed 30.57e70.27% inhibitory effect on 2-AA toward TA100 (Fig. 2). On the other hand, no inhibitory effect on 4-NP toward TA98 was noted with freeze-dried E. purpurea flower ethanolic extract (Fig. 2). Nevertheless, the freeze-dried E. purpurea flower ethanolic extract exhibited 0e9.91% inhibitory effect on NaN3 toward TA100. In the case of TA98, no inhibitory effect was shown by extract at 0.25e5.0 mg/plate. In the case of TA100, a weak inhibitory effect was shown by extract at 2.5e5.0 mg/plate, and it showed no inhibitory effects at 0.25e1.0 mg/plate. The freeze-dried E. purpurea flower ethanolic extract tended to inhibit the mutagenicity of indirect-acting mutagen that required metabolic activation of S9 mix more markedly than that of direct-acting mutagen. Some studies have been reported with possible explanation for the mechanism of protection against mutagens (Marnewick et al., 2000; Yen & Chen, 1995). Yen and Chen (1995) indicated that compounds with antioxidant activity could inhibit mutation and cancer due to the fact that they can scavenge a free radical or induce an antioxidant enzyme. Marnewick et al. (2000) reported that tea components could directly interact with the genotoxic reactive intermediates that may result in the prevention of mutagenesis. Furthermore, the flavanol structures in catechins provide a nucleophilic characteristic to react with an electrophilic mutagen to form flavanol-mutagen adducts which may prevent the occurrence of mutagenicity (Stich, 1991). In this study, freeze-dried E. purpurea flower ethanolic extract exhibits good scavenging effect on free radicals, reducing power and metal-binding ability. Thus, it appears that these relevant bioactive components and properties of freezedried E. purpurea flower ethanolic extract may contribute to antimutagenic potencies against different types of chemical mutagen. 4. Conclusion The extraction yield, total phenols, individual and total caffeic acid derivatives extracted from freeze-dried E. purpurea flowers were affected by ethanol/water ratio and extraction temperature. When the extraction yields of bioactive compounds were taken into consideration, the optimal extraction conditions of E. purpurea flowers were 50% ethanol and 65 C of extracting temperatures. The 50% ethanolic extract of E. purpurea flowers were good in antioxidant properties, and did not show toxicity and mutagenicity toward S. typhimurium test strains TA98 and TA100 with the concentrations that enabled antimutagenic activity. Furthermore, E. purpurea flowers ethanolic extract exhibits strong antimutagenicity when TA98 and TA100 were used as experimental microorganisms in the presence of metabolic activator. Acknowledgment The authors would like to thank National Science Council of Taiwan for financially supporting this research under Grant No. NSC-94-2214-E-241-002 and NSC-95-2221-E-241-002.
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References Alothman, M., Bhat, R., & Karim, A. A. (2009). Antioxidant capacity and phenolic content of selected tropical fruits from Malaysia, extracted with different solvents. Food Chemistry, 115, 785e788. Barnes, J., Anderson, L. A., Gibbons, S., & Phillipson, J. D. (2005). Echinacea species (Echinacea angustifolia (DC.) Hell., Echinacea pallida (Nutt.) Nutt., Echinacea purpurea (L.) Moench): a review of their chemistry, pharmacology and clinical properties. Journal of Pharmacy and Pharmacology, 57(8), 929e954. Bauer, R., & Wagner, H. (1991). Echinacea species as potential immunostimulating drugs. In H. Wagner, & N. R. Farnsworth (Eds.), Economic and medicinal plant research, Vol. 5 (pp. 253e321). New York: Academic Press. Caillet, S., Lessard, S., Lamoureux, G., & Lacroix, M. (2011). Umu test applied for screening natural antimutagenic agents. Food Chemistry, 124, 1699e1707. Camel, V. (2000). Microwave-assisted solvent extraction of environmental samples. Trends in Analytical Chemistry, 19, 229e248. Dinis, T. C. P., Madeira, V. M. C., & Almeida, L. M. (1994). Action of phenolic derivatives (acetaminophen, salicylate, and 5-amino salicylate) as inhibitors of membrane lipid peroxidation and as peroxyl radical scavengers. Archives of Biochemistry and Biophysics, 315, 161e169. Ebrahimzadeh, M. A., Pourmorad, F., & Bekhradnia, A. R. (2008). Iron chelating activity, phenol and flavonoid content of some medicinal plants from Iran. African Journal of Biotechnology, 7, 3188e3192. Hu, C., & Kitts, D. D. (2000). Studies on the antioxidant of Echinacea root extract. Journal of Agricultural and Food Chemistry, 48, 1466e1472. Ikken, Y., Morales, P., Martinez, A., Marin, M. L., Haza, A. I., & Cambero, M. I. (1999). Antimutagenic effect of fruit and vegetable ethanolic extracts against N-nitrosamines evaluated by the Ames test. Journal of Agricultural and Food Chemistry, 47, 3257e3264. Kim, S. J., Murthy, H. N., Hahn, E. J., Lee, H. L., & Paek, K. Y. (2007). Parameters affecting the extraction of ginsenosides from the adventitious roots of ginseng (Panax ginseng C.A. Meyer). Separation and Purification Technology, 56, 401e406. Lin, S. D., Sung, J. M., & Chen, C. L. (2011). Effect of drying and storage conditions on caffeic acid derivatives and total phenolics of Echinacea Purpurea grown in Taiwan. Food Chemistry, 125, 226e231. Marnewick, J. L., Wentzel, C. A., Gelderblom, C. A., & Joubert, E. (2000). An investigation on the antimutagenic properties of South African herbal teas. Mutation Research, 471, 157e166. Maron, D. M., & Ames, B. N. (1983). Revised methods for the Salmonella mutagenicity test. Mutation Research, 113, 173e215. Mengs, U., Clare, C. B., & Poiley, J. A. (1991). Toxicity of Echinacea purpurea. Acute, subacute and genotoxicity studies. Arzneimittel-Forschung/Drug Research, 41, 1076e1081. Mortelmans, K., & Zeiger, E. (2000). The Ames Salmonella/microsome mutagenicity assay. Mutation Research, 455, 29e60. Ong, T., Wong, W. Z., Stewart, J. D., & Brockman, H. E. (1986). Chlorophyllin: a potent antimutagen against environmental and dietary complex mixtures. Mutation Research, 173, 111e115. Oyaizu, M. (1986). Studies on products of browning reactions: antioxidative activities of products of browning reaction prepared from glucosamine. Japanese Journal of Nutrition, 44, 307e315. Pellati, F., Benvenuti, S., Magro, L., Melegari, M., & Soragni, F. (2004). Analysis of phenolic compounds and radical scavenging activity of Echinacea spp. Journal of Pharmaceutical and Biomedical Analysis, 35, 289e301. Perry, N. B., Burgess, E. J., & Glennie, V. A. (2001). Echinacea standardization: analytical methods for phenolic compounds and typical levels in medicinal species. Journal of Agriculture and Food Chemistry, 49, 1702e1706. Shahidi, F., & Wanasundara, P. K. J. (1992). Phenolic antioxidants. Critical Reviews in Food Science and Nutrition, 32, 67e103. Shimada, K., Fujikawa, K., Yahara, K., & Nakamura, T. (1992). Antioxidative properties of xanthan on the autoxidation of soybean oil in cyclodextrin emulsion. Journal of Agricultural and Food Chemistry, 40, 945e948. Spelman, K., Wetschler, M. H., & Cech, N. B. (2009). Comparison of alkylamide yield in ethanolic extracts prepared from dry Echinacea purpurea utilizing HPLC-ESIMS. Journal of Pharmaceutical and Biomedical Analysis, 49, 1141e1149. Spigno, G., & De Faveri, D. M. (2007). Antioxidants from grape stalks and marc: influence of extraction procedure on yield, purity and antioxidant power of the extracts. Journal of Food Engineering, 78, 793e801. Stanisavljevi c, I., Stoji cevi c, S., Veli ckovi c, D., Veljkovi c, V., & Lazi c, M. (2009). Antioxidant and antimicrobial activities of Echinaces (Echinacea purpurea L.) extracts obtained by classical and ultrasound extraction. Chinese Journal of Chemical Engineering, 17, 478e483. Stich, H. F. (1991). The beneficial and hazardous effects of simple phenolic compounds. Mutation Research, 259, 307e324. Stuart, D. L., & Wills, R. B. H. (2000). Factors affecting the extraction of alkylamides and cichoric acid during ethanolic processing of Echinacea purpurea (L.) Moench. Australian Journal of Experimental Agriculture, 40, 873e877. Taiwan Food and Drug Administration. (2011). Echinacea purpurea. http://consumer. fda.gov.tw/Food/Material.aspx?nodeID¼160#. Thygesen, L., Thulinn, J., Mortensen, A., Skibsted, L. H., & Molgaard, P. (2007). Antioxidant activity of cichoric acid and alkamides from Echinacea purpurea, alone and in combination. Food Chemistry, 101, 74e81. Waleh, N. S., Rapport, S. J., & Mortelmans, K. E. (1982). Development of a toxicity test to be coupled to the Ames Salmonella assay and the method of construction of the required strains. Mutation Research, 97, 247e256.
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Y.-L. Tsai et al. / LWT - Food Science and Technology 46 (2012) 169e176
Wang, L., & Weller, C. L. (2006). Recent advances in extraction of nutraceuticals from plants. Trends in Food Science and Technology, 17, 300e312. Wu, C. H., Murthy, H. N., Hahn, E. J., Lee, H. L., & Paek, K. Y. (2008). Efficient extraction of caffeic acid derivatives from adventitious roots of Echinacea purpurea. Czech Journal of Food Sciences, 26, 254e258. Xi, J., Shen, D., Zhao, S., Lu, B., Li, Y., & Zhang, R. (2009). Characterization of polyphenols from green tea leaves using a high hydrostatic pressure extraction. International Journal of Pharmaceutics, 382, 139e143. Yam, T. S., Shah, S., & Hamilton-Miller, J. M. T. (1997). Microbiology activity of whole and fractionated crude extracts of tea (Camellia sinensis), and of tea components. FEMS Microbiology Letters, 152, 169e174.
Yamaguchi, R., Tatsumi, M. A., Kato, K., & Yoshimitsu, U. (1988). Effect of metal salts and fructose on the autoxidation of methyl linoleate in emulsions. Agricultural and Biological Chemistry, 52, 849e850. Yen, G. C., & Chen, H. Y. (1995). Antioxidant activity of various tea extracts in relation to their antimutagenicity. Journal of Agricultural and Food Chemistry, 43, 27e32. Zahin, M., Aqil, F., & Ahmad, I. (2010). Broad spectrum antimutagenic activity of antioxidant active fraction of Punica granatum L. peel extracts. Mutation Research, 703, 99e107. Zhai, Z., Haney, D., Wu, L., Solco, A., Murphy, P. A., Wurtele, E. S., et al. (2007). Alcohol extracts of Echinacea inhibit production of nitric oxide and tumor necrosis factoralpha by macrophages in vitro. Food Agriculture Immunology, 18, 221e236.