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Variation of active constituents and antioxidant activity in pyrola [P. incarnata Fisch.] from different sites in Northeast China
Food Chemistry 141 (2013) 2213–2219
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Variation of active constituents and antioxidant activity in pyrola [P. incarnata Fisch.] from different sites in Northeast China Dong-Yang Zhang, Meng Luo, Wei Wang, Chun-Jian Zhao, Cheng-Bo Gu, Yuan-Gang Zu, Yu-Jie Fu ⇑, Xiao-Hui Yao, Ming-Hui Duan State Engineering Laboratory for Bio-Resource Eco-Utilization, Northeast Forestry University, Harbin 150040, PR China Engineering Research Center of Forest Bio-Preparation, Ministry of Education, Northeast Forestry University, Harbin 150040, PR China
a r t i c l e
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Article history: Received 20 February 2013 Received in revised form 10 May 2013 Accepted 13 May 2013 Available online 21 May 2013 Keywords: Pyrola Total phenolic content Antioxidant activity HPLC Principal component analysis
a b s t r a c t The variation of antioxidant activity and active components in pyrola [Passiflora incarnata Fisch.] from eight sites in Northeast China were investigated. Total phenolic and flavonoid contents were determined and varied within the range of 39.66–181.48 mg/g and 2.47–22.11 mg/g, respectively. Antioxidant activities were determined by scavenging activity against DPPH and ABTS, by a reducing power test and by a b-carotene-linoleic acid bleaching test. The IC50 of Tahe samples determined by the DPPH test was 0.106 ± 0.006 mg/mL which was very close to that of Vc (0.076 ± 0.004 mg/mL). The Tahe samples had good antioxidant activity. Principal component activity analysis indicated that the Tahe samples of P. incarnata had the highest potential antioxidant properties, and may be a valuable antioxidant natural resource in the northeast of China. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction An increasing number of studies indicate that many kinds of plants can reduce the risk of chronic degenerative diseases such as cardiovascular disease, cancers, and others. The associated beneficial effects have been attributed to various components which are regarded as antioxidants including phenolics, flavonoids, carotenoids and tocopherols. Oxygen is essential to many living organisms for producing energy to supply biological processes. However, about 2–3% of oxygen taken into the human body is converted to reactive oxygen species (ROS) and free radicals, which enhance oxidative damage to various biomolecules of DNA, proteins, small cellular molecules and membrane lipids (Dai & Rabinovitch, 2009; Lu, Lin, Yao, & Chen, 2010; Shen, 2010). This has driven researchers to search for a link between reduced risk of degenerative diseases and dietary-related antioxidant intake via prevention of cellular oxidative damage (Thaipong, Boonprakob, Crosby, Cisneros-Zevallos, & Byrne, 2006). The measurement of the antioxidant activity of food products and ingredients is a matter of growing interest. In addition to the presumed association with health benefits, antioxidants may inhibit, retard or ameliorate oxidative spoilage (Serrano, Goni, & Saura-Calixto, 2007; Tsoukalas, Katsanidis, Marantidou, & Bloukas, 2011; Zulueta, Esteve, & Frigola, 2009). Determination of the total ⇑ Corresponding author. Tel./fax: +86 451 82190535. E-mail address: [email protected] (Y.-J. Fu). 0308-8146/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2013.05.045
antioxidant capacity cannot be performed accurately by any single method due to the diversity of phytochemicals present and the associated chemical moiety diversity (Chu, Chang, & Hsu, 2000). Consequently, at least two different methods should be used to evaluate antioxidant potential capacity. Pyrola, a perennial evergreen herbaceous plant, is mainly distributed in the northern hemisphere in temperate and cold temperate regions around the world. Pyrolae (Chinese name: Lu xian cao, Lu han cao, or Lu ti cao), the whole herb of Pyrola calliantha H. Andr., Passiflora incarnata Fisch., Pyrola decorate H. Andr., or other Pyrola plants (Pyrolaceae), has been used in tonics, sedatives, analgesics against rheumatoid arthritis, and hemostatics (Klyomi et al., 1992). Nowadays, because of its function of slowing down ageing and boosting immunity, it was used as a kind of tea called Lu shou cha for daily drinking in China (Sun et al., 2011). Pyrola is widespread as an edible plant for food industry in landscape area (National Commission of Chinese Pharmacopoeia, 2005). The water extract of the plant was reported to inhibit the growth of many kinds of human pathogenic bacilli in vitro, it can be used in refreshing foods (Dictionary of Chinese Traditional Medicine, 1986). P. incarnata is mainly distributed in the northeast of China. Its’ main constituents are chimaphilin, arbutin, epicatechin, catechin, 200 -O-galloylhyperin, hyperin quercetin, pyrolatin and other naphthoquinones (Kaqawa et al., 1992; Kosuge et al., 1985; Li et al., 2008; Yazaki, Shida, & Okuda, 1989). The main distribution sites of P. incarnata in the northeast of China were Shengshan, Jiagedaqi, Tahe, Wuying, Xinlin, Wudalianchi and Tulihe. It has been well
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known that the content varies in its’ active ingredient, and is affected by environmental, cultural, and post-harvesting conditions. So it is important to evaluate the quality of P. incarnata from different sites in the northeast of China. This variation may aid harvest and the development of P. incarnata as a potential antioxidant resource. Contents of active compounds in P. incarnata samples collected from the above 8 different sites was analysed in this study. The total phenolic contents, total flavonoid contents and the antioxidant activities including tests of scavenging activity, reducing power and b-carotene-linoleic acid bleaching were also determined. The principal component analysis (PCA) was conducted to detect cluster formation and establish relationships among different site samples, active compounds and antioxidant properties. 2. Materials and methods 2.1. Chemicals 1,1-diphenyl-2-picrylhydrazyl (DPPH), b-carotene, linoleic acid, Tween 40, Folin–Ciocalteu reagent, standards of gallic acid, rutin, arbutin, epicatechin, catechin, hyperin, 200 -O-galloylhyperin, quercitrin, pyrolatin, chimaphilin, butylated hydroxytoluene (BHT) and ascorbic acid (Vc) were all purchased from Delta (Wuhu, China). A total antioxidant capacity assay kit (ABTS assay) obtained from Beyotime Institute of Biotechnology (China). Methanol of chromatographic grade was purchased from J & K Chemical Ltd. (China). Formic acid was chromatographic grade (Dima Technology INC., USA). Ethanol was of analytical grade and purchased from Tianjin Chemical Reagents Co. (Tianjing, China). Deionised water was purified by a Milli-Q Water Purification system (Millipore, MA, USA). For standard solutions, appropriate amounts of reference compounds were dissolved in methanol to yield the stock solutions at a concentration of 1 mg/mL, respectively. All solutions prepared for HPLC were filtered through 0.45 lm nylon membranes before used. 2.2. Plant material P. incarnata Fisch. samples were collected in summer from the northeast of China from eight sites (Fig. 1): Tahe forest farm, Xinlin
forest farm, Tulihe forest farm, Jiagedaqi forest farm, Shengshan forest farm, Dayangshu forest farm, Wudalianchi forest farm and Wuying forest farm. The main climate data were shown in Table S1. The samples were identified by Professor Shaoquan Nie from the Key Laboratory of Forest Plant Ecology, Ministry of Education, Northeast Forestry University, PR China. Voucher specimens were deposited in the herbarium of this Key Laboratory. The leaves were naturally dried. After that, the dried leaves were pulverised, and then stored in a dry place at room temperature until used. 2.3. Sample extraction Each samples (10 g) were extracted three times under sonication in an ultrasonic bath (Kunshan Ultrasonic Instrument, Jiangsu, China) with 80% (v/v) aqueous ethanol (1.5 L) at 40 °C for 40 min. The extract liquor was combined three times and concentrated by a rotary evaporator (RE-52AA, Shanghai Huxi Instrument Co., China) to obtain the crude extract. 2.4. Determination of total phenolic and flavonoid contents 2.4.1. The amounts of total phenolics Total phenolic contents of each sample were determined by using the Folin–Ciocalteu method according to Wu et al. (2010) with slight modifications. Briefly, 40 lL of blank and each extract/fractions were mixed with 1.8 mL of Folin–Ciocalteu reagent (10-fold diluted with deionised water). The mixture was allowed to stand for 5 min at room temperature before it was neutralised with the addition of 1.2 mL of 7.5% sodium carbonate solution. After 1.5 h of reaction at ambient temperature and in darkness, the absorbance at 765 nm was measured using spectrophotometer (UNICO, Shanghai, China) and the phenolic contents were calculated using gallic acid as a standard. The total phenolic contents were expressed in milligramme gallic acid equivalents (mg GAE/g extract). All the measurements were taken in triplicate. 2.4.2. The amounts of total flavonoids The flavonoid contents were determined using an aluminium chloride method with rutin as a reference compound. This method is based on the formation of a flavonoid-aluminium complex with an absorptivity maximum at 410 nm (Chang, Yang, Wen, & Chern, 2002). About 0.5 mL of each extract/fractions in 50% ethanol (4 mg/ mL) was added to a 10 mL volumetric flask containing 3 mL of
Fig. 1. Different collection area of pyrola [P. incarnata] in the northeast of China.
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0.1 M potassium acetate (CH3COOK) in deionised water and 2 mL of 0.1 M aluminium chloride hexahydrate (AlCl3). After incubation at room temperature for 20 min, then immediately metered volume to 10 mL with 50% ethanol, the reaction mixture absorbance was measured at 410 nm. Blank sample was used 500 lL of 50% ethanol instead of 500 lL of samples. Three replicates were made for each test sample. The total flavonoid content was expressed as rutin equivalents, in mg/g extract. 2.5. DPPH radical scavenging activity assay The free radical-scavenging activities of samples were measured in terms of hydrogen donating or radical-scavenging ability using the stable radical DPPH (Wu et al., 2009). The different concentrations of the samples in 50% ethanol (100 lL) were mixed with 50% ethanol (1.4 mL) and then added to 0.004% DPPH (1 mL, Sigma–Aldrich) in ethanol. The mixture was shaken vigorously and then immediately incubated in darkness. After 70 min, the reaction reached a plateau. The decreasing of the DPPH solution absorbance was determined in a UV–Vis spectrophotometer (UNICO, Shanghai, China) to monitor absorbance at 517 nm. Ascorbic acid (Sigma–Aldrich), a stable antioxidant, was used as a positive reference. The DPPH radical-scavenging activity in percentage of sample was calculated as follows: DPPH scavenging activity (%) = (1 A517 sample/A517 DPPH solution) 100. 2.6. ABTS radical scavenging activity assay The total antioxidant capacity was evaluated using the ABTS method (Beyotime Institute of Biotechnology, China). ABTS radical cation (ABTS+) solution was produced by reacting ABTS stock solution with 2.45 mmol/L potassium persulfate in the dark at room temperature for 12–16 h before use, and then the resulting ABTS+ solution was diluted with 80% ethanol to adjust the absorbance to 0.70 ± 0.05 at 734 nm. After addition of 10 lL of sample or trolox standard to 200 lL of diluted ABTS+ solution, absorbance at 734 nm was measured after 2–6 min in the dark at room temperature. Trolox, a water-soluble analogue of vitamin E, is used as the reference standard to prepare a calibration curve for a concentration range of 0.15–1.5 mM. Results were expressed as mmol/g Trolox equivalent antioxidant capacity (TEAC). 2.7. The reducing power The reducing power was measured according to the method of Wu et al. (2010) with some modification. An aliquot of each sample (0.5 mL), with different concentrations, was mixed with 0.5 mL of phosphate buffer (0.2 M, pH 6.6) and 0.5 mL of 1% potassium ferricyanide [K3Fe(CN)6]. The reaction mixture was incubated at 50 °C for 20 min. After incubation, 0.5 mL of 10% trichloroacetic acid (TCA) was added, followed by centrifugation at 650 xg for 10 min. The supernatant (0.5 mL) was mixed with 0.5 mL of distilled water and 0.1 mL of 0.1% ferric chloride (FeCl3). The absorbance of all sample solutions was measured at 700 nm. An increased absorbance indicated increased reducing power. BHT was used as the positive control. 2.8. b-carotene-linoleic acid bleaching assay The lipid antioxidant activities of the samples were determined using b-carotene-linoleic acid bleaching assay (Wu et al., 2010). A solution of b-carotene was prepared by dissolving b-carotene (10 mg) in chloroform (10 mL). The b-carotene-chloroform solution (0.2 mL) was pipetted into a round-bottomed flask containing linoleic acid (20 mg) and Tween 40 emulsifier (200 mg). Chloroform was removed using a rotary evaporator at 40 °C for 5 min,
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and distilled water (50 mL) was added to the flask slowly with vigorous agitation to form an emulsion. Aliquots (5 mL) of this emulsion were added into different test tubes containing different concentrations of sample solutions (0.2 mL), and the absorbance was immediately measured at 470 nm against a blank, consisting of an emulsion without b-carotene. The tubes were incubated in a water bath at 50 °C, and the oxidation of the emulsion absorbance was measured at 470 nm over a 60 min period by a spectrophotometer. Control samples contained 200 lL of water instead. BHT, a stable antioxidant, was used as a synthetic reference. The antioxidant activity was expressed as inhibition percentage with reference to the control after a 60 min incubation using the following equation: AA = 100(DRC – DRS)/DRC, where AA = antioxidant activity, DRC = degradation rate of the control = [ln(a/b)/60], DRS = degradation rate in presence of the sample = [ln(a/b)/60], a = absorbance at time 0, and b = absorbance at 60 min.
3. Results and discussion 3.1. Total phenolic and flavonoid contents Phenolic compounds are considered as a major group of compounds that contributed to the antioxidant activity. In order to understand the relationship between antioxidant activity and total phenolic content (TPC), the TPC of P. incarnata samples from 8 different sites in the northeast of China were determined using the Folin–Ciocalteu phenol reagent. The results are expressed in gallic acid equivalents (GAE) and presented in Table 1. The TPC of Tahe and Tulihe were 181.48 ± 3.71 GAE mg/g and 175.09 ± 2.28 GAE mg/g, which were much higher than those of other samples. The lowest TPC was in Shengshan which was only 39.66 ± 1.37 GAE mg/g. The TPC is arranged as following sequence: Tahe > Tulihe > Wudalianchi > Xinlin > Wuying > Dayangshu > Jiagedaqi > Shengshan. Flavonoids are the most commonly and widely distributed group of plant phenolic compounds, which are usually very effective antioxidants. The total flavonoid contents (TFC) of P. incarnata Fisch. from eight different sites in the northeast of China were evaluated by aluminium chloride spectrophotometer. Using rutin as a standard (y = 2.5881x0.0051; r2 = 0.9997). The data (Table 1) show that the highest and lowest flavonoid contents were observed from samples of Tahe and Shengshan, 22.11 ± 1.25 and 2.47 ± 0.34 RE mg/g, respectively. TFC of the Jiagedaqi sample was the second lowest which was only higher than that of Shengshan. TFC is arranged as following sequence: Tahe > Tulihe > Wudalianchi > Xinlin > Wuying > Dayangshu > Jiagedaqi > Shengshan, varied from 2.47 ± 0.34 to 22.11 ± 1.25 RE mg/g. These results were very similar to the results of TPC. TPC and TFC of Tahe were higher than those of other sites. Tahe is located at a high latitude site (Fig. 1) that has the lowest annual average temperature and annual average precipitation among the eight sites. Some literature reports indicate that low temperature stress and stress from water shortage may enhance secondary metabolite content and antioxidant activity (Ill et al., 2006; Zhu, Liang, Han, & Wang, 2009). Low temperature stress may cause damage to cells by inducing reactive oxygen species (ROS) by disrupting the scavenging systems that quench oxygen. And the synthesis of certain phenolic compounds can occur in response to the low temperature stress (Pennycooke, Cox, & Stushnoff, 2005). Water shortage stress may decrease the consumed content of NADPH + H+ within the Calvin cycle. The excess NADPH + H+ is used for the synthesis of secondary metabolites which result in the increase of TPC (Selmar & Kleinwächter, 2013). Thus, the highest TPC and TFC of Tahe may be due to the low annual average temperature and low annual average precipitation. A similar
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Table 1 Total phenolics, flavonoids and antioxidant activity (DPPH, ABTS, reducing power and anti-b-carotene bleaching assay) of different pyrola samples from Northeast China. Sample
Total phenolics (GAE mg/g)
Total flavonoids (RE mg/g)
DPPH IC50a (mg/mL)
ABTSb (mmol/g Trolox)
Reducing power IC50a (mg/mL)
Anti-b-carotene bleaching IC50a (mg/mL)
Shengshan Jiagedaqi Tahe Wuying Xinlin Wudalianchi Tulihe Dayangshu Vc BHT
39.66 ± 1.37 77.76 ± 2.11 181.48 ± 3.71 100.67 ± 6.88 125.52 ± 3.00 145.99 ± 2.44 175.09 ± 2.28 96.47 ± 3.17 – –
2.47 ± 0.34 6.66 ± 0.50 22.11 ± 1.25 10.77 ± 0.83 13.69 ± 0.45 15.46 ± 0.61 19.23 ± 0.65 11.91 ± 1.62 – –
0.282 ± 0.019 0.187 ± 0.010 0.106 ± 0.006 0.160 ± 0.082 0.134 ± 0.010 0.124 ± 0.007 0.112 ± 0.004 0.148 ± 0.015 0.076 ± 0.004 ND
0.254 ± 0.013 0.474 ± 0.020 0.611 ± 0.016 0.521 ± 0.022 0.564 ± 0.043 0.584 ± 0.012 0.643 ± 0.079 0.532 ± 0.017 1.057 ± 0.095 ND
0.318 ± 0.020 0.161 ± 0.018 0.083 ± 0.014 0.162 ± 0.013 0.127 ± 0.008 0.138 ± 0.007 0.102 ± 0.011 0.123 ± 0.010 ND 0.141 ± 0.018
0.089 ± 0.004 0.097 ± 0.004 0.038 ± 0.005 0.042 ± 0.003 0.032 ± 0.004 0.055 ± 0.007 0.061 ± 0.004 0.067 ± 0.004 ND 0.017 ± 0.001
Each value is expressed as the mean ± standard deviation (n = 3). ND, not determined. a IC50 value: the concentration at which the antioxidant activity inhibition ratio reached 50%; 1,1-diphenyl-2-picrylhydrazyl (DPPH) was scavenged by 50%; the absorbance was 0.5 for reducing power; and b-carotene bleaching was inhibition ratio reached 50%. b ABTS were expressed as mmol/g Trolox equivalent antioxidant capacity (TEAC).
Fig. 2. DPPH radical-scavenging activities (A), ABTS radical-scavenging activities (B), reducing power (C), anti-b-carotene bleaching (D) of different pyrola samples in the northeast of China. Each value is expressed as mean ± standard deviation (n = 3).
phenomenon may occur at Tulihe which also has lower annual average temperatures and lower annual average precipitation. 3.2. Antioxidant activity 3.2.1. DPPH radical scavenging activity assay The relatively stable organic radical DPPH has been widely used for the determination of antioxidant activity of pure antioxidant compounds as well as different plant extracts. For evaluation of antioxidant activity of P. incarnata different samples were measured and compared with their DPPH radical scavenging activities. Fig. 2A illustrates the DPPH radical scavenging activities of the different P. incarnata samples. Among the results of DPPH scavenging activities, the Tahe sample had the highest DPPH radical scavenger activity followed by Tulihe, Wudalianchi, Xinlin, Dayangshu,
Wuying, Jiagedaqi and Shengshan. In order to quantify the antioxidant activity further, the IC50 was calculated (Table 1). The lowest IC50 of Tahe was 0.106 ± 0.006 mg/mL reflecting the highest DPPH radical scavenging activity among all samples. The IC50 values of Tulihe, Wudalianchi, Xinlin and Dayangshu were also high, which were only slightly lower than the Tahe samples. From Fig. 1, it can also be seen that when the concentration of samples was 0.25 mg/mL, the DPPH radical scavenging activities of Tahe and Tulihe were more than 85% which were very close to that of Vc indicating that samples from Tahe and Tulihe had the most potent antioxidant activity. 3.2.2. ABTS radical scavenging activity assay The ABTS radical scavenging activity assay is applied to measure the total antioxidant capacity of samples. The results of ABTS
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D.-Y. Zhang et al. / Food Chemistry 141 (2013) 2213–2219 Table 2 The content of the active compounds in different pyrola samples. Sample
Arbutin (mg/g)
Epicatechin (mg/g)
Catechin (mg/g)
Hyperin (mg/g)
20 0 -O-galloyl-hyperin (mg/g)
Pyrolatin (mg/g)
Quercetin (mg/g)
Chimaphilin (mg/g)
Shengshan Jiagedaqi Tahe Wuying Xinlin Wudalianchi Tulihe Dayangshu
0.648 ± 0.047 1.975 ± 0.140 1.695 ± 0.235 2.300 ± 0.132 2.655 ± 0.199 1.875 ± 0.141 2.605 ± 0.178 1.865 ± 0.174
0.108 ± 0.008 0.466 ± 0.038 2.310 ± 0.174 1.053 ± 0.074 1.127 ± 0.014 1.040 ± 0.120 1.810 ± 0.123 1.066 ± 0.089
– 0.120 ± 0.026 0.176 ± 0.010 0.083 ± 0.007 0.081 ± 0.010 0.035 ± 0.003 0.048 ± 0.002 0.050 ± 0.003
0.640 ± 0.033 0.838 ± 0.038 2.742 ± 0.153 1.719 ± 0.085 1.756 ± 0.425 1.771 ± 0.666 2.304 ± 0.228 1.554 ± 0.088
2.300 ± 0.257 3.656 ± 0.236 8.839 ± 0.226 6.331 ± 0.150 6.568 ± 0.197 5.720 ± 0.263 7.423 ± 0.621 6.612 ± 0.335
– – 0.318 ± 0.015 0.124 ± 0.007 – 0.179 ± 0.001 0.251 ± 0.001 –
0.032 ± 0.002 – 0.046 ± 0.002 0.039 ± 0.002 0.047 ± 0.003 0.050 ± 0.003 – –
0.084 ± 0.003 0.172 ± 0.001 0.081 ± 0.008 0.468 ± 0.002 0.416 ± 0.042 0.116 ± 0.001 0.232 ± 0.006 0.405 ± 0.025
radical scavenging activity assay are expressed as trolox equivalent antioxidant capacity (TEAC). In Fig 2B, the highest ABTS+ scavenging activity was found for Tulihe which was differed from the results of the DPPH radical scavenging activity assay. But compared with Tulihe, the TEAC of Tahe and Wudalianchi were not much lower. The TEAC of Shengshan was 0.254 ± 0.013 mmol/g which was still the lowest among the eight samples. The ABTS radical scavenging activity values decreased in the order of Tulihe > Tahe > Wudalianchi > Xinlin > Dayangshu > Wuying > Jiagedaqi > Shengshan. Compared with their DPPH free radical scavenging activities, the trend for ABTS radical scavenging activity did not vary markedly. The TEAC of Vc was 1.057 ± 0.095 (Table 1) which was much higher than that of the P. incarnata samples. Thus, there is a wide gap between Vc and P. incarnata extracts for ABTS radical scavenging activity.
3.2.3. The reducing power It has been reported that reducing power is associated with antioxidant activity and may serve as a significant reflection of the antioxidant activity (Oktay, Gülçin, & Küfreviog˘lu, 2003). Compounds with reducing power indicate that they are electron donors, and can reduce the oxidised intermediates of lipid peroxidation processes, so that they can act as primary and secondary antioxidants (Yen & Chen, 1995). As shown in Fig. 2C, various concentrations of each sample were used for this assay and all of the samples exhibited reducing power. The samples of Tahe, Tulihe, Dayangshu, Xinlin and Wudalianchi revealed good reducing power, with the IC50 of 0.083 ± 0.014, 0.102 ± 0.011, 0.123 ± 0.010, 0.127 ± 0.008 and 0.138 ± 0.007 mg/mL, respectively. The data of the positive control (BHT) was 0.141 ± 0.018 mg/mL. This indicated that the Tahe sample had a remarkable antioxidant capacity better than that of BHT. The Dayangshu sample had better reducing power than that of Xinlin and Wudalianchi although its DPPH and ABTS radical scavenging activity were lower than Xinlin and Wudalianchi.
3.2.4. b-carotene-linoleic acid bleaching assay The b-carotene-linoleic acid bleaching inhibition effect of BHT and eight P. incarnata samples are shown in Fig. 2D. According to Fig. 2D, all the test samples inhibited the oxidation of linoleic acid, with increasing concentration. The IC50 of the eight P. incarnata samples are revealed in Table 1. The results obtained by b-carotene-linoleic acid bleaching inhibition test were different from those of the DPPH method and ABTS method. The Xinlin sample had the best b-carotene-linoleic acid bleaching activity which was very different to the results of DPPH and ABTS test. When the concentration of Xinlin was 0.5 mg/mL, the inhibition could reach to 93.14%. The b-carotene-linoleic acid bleaching activities of Tahe and Xinlin were also relative high. The Tulihe sample which had good DPPH and ABTS radical scavenging activity, had lower b-carotene-linoleic acid bleaching than most of the samples.
All the samples possessed b-carotene-linoleic acid bleaching activities (Fig. 2D, Table 1). 3.3. Constituents comparison of P. incarnata from different sites The constituents of the P. incarnata samples were analysed by HPLC and the data of the 8 principal compounds in plant extracts are presented in Table 2. The most abundant phenolics found in the P. incarnata samples were arbutin, epicatechin, hyperin and 200 -O-galloylhyperin. Other phenolic compounds catechin, pyrolatin, quercitrin and chimaphilin were found at lower concentrations. Not all of the active compounds were detected in the P. incarnata samples, as in catechin in Shengshan, pyrolatin in Shengshan, Jiagedaqi, Xinlin and Dayangshu and quercetin in Jiagedaqi, Tulihe and Dayangshu (Table 2). The highest content of arbutin was in Xinlin and Tulihe followed by Wuying, Jiagedaqi, Wudalianchi, Dayangshu, Tahe and Shengshan. The most abundant component was 200 -O-galloylhyperin ranging from 2.300 ± 0.257 to 8.839 ± 0.226 mg/g. Another higher content compound was hyperin ranged from 0.640 ± 0.033 to 2.742 ± 0.153 mg/g. The highest contents of 2’’-O-galloylhyperin and hyperin, found in the Tahe samples were 8.839 ± 0.226 and 2.742 ± 0.153 mg/g, respectively. Meanwhile, the sample in Tulihe also had high amounts of 200 -Ogalloylhyperin and hyperin. Because of the high latitude, the annual average temperature and precipitation in Tahe is lower, and for Tulihe, its geographic location is in Greater Khingan which can also provide a cold environment for the accumulation of phenolic compounds. The annual average precipitation of Tahe and Tulihe are also smaller (Table S1), drought may induce the accumulation of more phenolic compounds. From the results of Table 1, 200 -O-galloylhyperin could be regard as the main active compound based on content, which is higher than other compounds. According to other research, 200 -O-galloylhyperin has anti-inflammatory, cough, blood pressure, and lower cholesterol, the cardiovascular and cerebrovascular protective role (Kaqawa et al., 1992; Lou, Yang, Song, & Wang, 2004). Thus, P. incarnata Fisch. is an important natural plant resource of 200 -O-galloylhyperin. Hyperin is a famous human health antioxidant in the world, it has been commercially extracted from Hypericum perforatum L., which has faced a big question of resource shortage in recent years. Thus, it is important to find a new resource rich in hyperin. The present result showed that P. incarnata contains abundant hyperin, and could be a potential resource for hyperin. Additionally, the components content of Tahe samples is obviously higher than those of other sites. Thus the Tahe site with low annual average temperature and low annual average precipitation suggests this cool climate site is suitable for the production of antioxidant rich P. incarnata. 3.4. Principal component analysis Principal component analysis (PCA) was performed to understand the interrelationships among the measured antioxidant
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Fig. 3. Principal component analysis loading plot of TPC, TFC, antioxidant activity and active compounds from of different pyrola samples in the northeast of China (A), Principal component analysis score plot of different pyrola samples in the northeast of China (B). RP, reducing power; AC, anti-b-carotene bleaching assay; GH, 200 -O-galloylhyperin.
activity, TPC, TFC and active compounds content. The results of PCA are shown in Fig. 3A. Two principal components, explaining the 80.4% of the total data variance, have been chosen on the basis of their eigenvalues (>1). PC 1 = 0.955 TPC + 0.972 TFC – 0.961 DPPH + 0.947 ABTS – 0.922 RP – 0.770 AC + 0.667 arbutin + 0.951 epicatechin + 0.557 catechin + 0.967 hyperin + 0.966 GH + 0.763 pyrolatin + 0.193 quercetin + 0.149 chimaphilin. PC 2 = 0.195 TPC + 0.175 TFC + 0.153 DPPH – 0.195 ABTS + 0.232 RP + 0.095 AC – 0.649 arbutin + 0.190 epicatechin + 0.130 catechin + 0.168 hyperin – 0.081 GH + 0.547 pyrolatin + 0.398 quercetin – 0.897 chimaphilin.
The first principal component (PC1) correlated well with TPC, TFC, DPPH, ABTS, reducing power, epicatechin, hyperin and 200 -Ogalloylhyperinwith the loadings 0.955, 0.972, 0.961, 0.947, 0.922, 0.951, 0.967 and 0.966, respectively. The second principal component (PC 2) was related to chimaphilin with loadings of 0.897. There were good correlations between TPC, TFC, DPPH and ABTS and these were consistent with the results in Fig 3A. Moreover, DPPH radical scavenging activity, b-carotene-linoleic acid bleaching assay, and reducing power were found to be similarly loaded on PC 1, which indicated the three properties were closely related to antioxidant activity. TPC and TFC also had high loading on PC1, which suggested phenolic compounds were good
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antioxidants. For PC 2, only chimaphilin get high loadings, while the antioxidant activity (DPPH radical scavenging activity, ABTS radical scavenging activity, b-carotene-linoleic acid bleaching assay, and reducing power) had low loading, which indicated that chimaphilin might not have good antioxidant activity. The content of 200 -O-galloylhyperin which correlated well with DPPH radical scavenging activity, ABTS radical scavenging activity and b-carotene-linoleic acid bleaching assay, was higher than other compounds. Thus, 200 -O-galloylhyperin can be regard as the main antioxidant component in P. incarnata Fisch. On the other hand, epicatechin, catechin and hyperin also has good antioxidant activity from some other reports (Rusak, Komes, Likic, Horzˇic´, & Kovacˇ, 2008; Xing et al., 2011) and this is also similar to our correlation analysis results. PCA can also provide an overview of the similarities and differences among the 8 P. incarnata samples from different areas. The Tahe sample found in the upper right part of Fig. 3B, which was positively correlated with PC1 and PC2. This sample can be best described as having high antioxidant activity and a high content of total phenolics and flavonoids. Close relationships were observed among Wuying, Dayangshu and Xinlin which indicated that they have similar antioxidant activity and contents of total phenolic and flavonoid. While the sample of shengshan had the largest negative score of PC 1, it indicated that shengshan exhibited the lowest antioxidant activity and contents of total phenolic and flavonoid.
4. Conclusions Pyrola [P. incarnata], grown in Northeast China, is a potentially rich source of desirable health attributes. The Tahe sample has high total phenolic and flavonoid contents and good antioxidant activity. Its’ IC50 of reducing power test is 0.083 ± 0.014 mg/mL which is better than that of BHT (0.141 ± 0.018 mg/mL) and the IC50 (0.106 ± 0.006 mg/mL) of DPPH test is also close to that of Vc (0.076 ± 0.004 mg/mL). These results indicate that the pyrola of Tahe possesses remarkable antioxidant activity. Moreover, eight compounds of different P. incarnata samples were quantified. 200 -O-galloylhyperin and hyperin were the most abundant components, and P. incarnata can be regarded as a potential natural plant resource of these two compounds. The Tahe sample also has a high content of active compounds including 200 -O-galloylhyperin, hyperin, arbutin and epicatechin. PCA indicates that P. incarnata from Tahe has the best quality, it could be a valuable antioxidant natural source in the northeast of China. The present study supports meaningful information for the collection and application of the P. incarnata resource in food production.
Acknowledgements The authors gratefully acknowledge the financial supports by Program for Agricultural Science and Technology Achievements Transformation Fund Program (2012GB23600641), Special Fund of Forestry Industrial Research for Public Welfare of China (201004040), Importation of International Advanced Forestry Science and Technology, National Forestry Bureau (2012-4-06) and Heilongjiang Province Science Foundation for Excellent Youths (JC200704).
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.foodchem.2013. 05.045.
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Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Variation of active constituents and antioxidant activity in pyrola [P. incarnata Fisch.] from different sites in Northeast China Dong-Yang Zhang, Meng Luo, Wei Wang, Chun-Jian Zhao, Cheng-Bo Gu, Yuan-Gang Zu, Yu-Jie Fu ⇑, Xiao-Hui Yao, Ming-Hui Duan State Engineering Laboratory for Bio-Resource Eco-Utilization, Northeast Forestry University, Harbin 150040, PR China Engineering Research Center of Forest Bio-Preparation, Ministry of Education, Northeast Forestry University, Harbin 150040, PR China
a r t i c l e
i n f o
Article history: Received 20 February 2013 Received in revised form 10 May 2013 Accepted 13 May 2013 Available online 21 May 2013 Keywords: Pyrola Total phenolic content Antioxidant activity HPLC Principal component analysis
a b s t r a c t The variation of antioxidant activity and active components in pyrola [Passiflora incarnata Fisch.] from eight sites in Northeast China were investigated. Total phenolic and flavonoid contents were determined and varied within the range of 39.66–181.48 mg/g and 2.47–22.11 mg/g, respectively. Antioxidant activities were determined by scavenging activity against DPPH and ABTS, by a reducing power test and by a b-carotene-linoleic acid bleaching test. The IC50 of Tahe samples determined by the DPPH test was 0.106 ± 0.006 mg/mL which was very close to that of Vc (0.076 ± 0.004 mg/mL). The Tahe samples had good antioxidant activity. Principal component activity analysis indicated that the Tahe samples of P. incarnata had the highest potential antioxidant properties, and may be a valuable antioxidant natural resource in the northeast of China. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction An increasing number of studies indicate that many kinds of plants can reduce the risk of chronic degenerative diseases such as cardiovascular disease, cancers, and others. The associated beneficial effects have been attributed to various components which are regarded as antioxidants including phenolics, flavonoids, carotenoids and tocopherols. Oxygen is essential to many living organisms for producing energy to supply biological processes. However, about 2–3% of oxygen taken into the human body is converted to reactive oxygen species (ROS) and free radicals, which enhance oxidative damage to various biomolecules of DNA, proteins, small cellular molecules and membrane lipids (Dai & Rabinovitch, 2009; Lu, Lin, Yao, & Chen, 2010; Shen, 2010). This has driven researchers to search for a link between reduced risk of degenerative diseases and dietary-related antioxidant intake via prevention of cellular oxidative damage (Thaipong, Boonprakob, Crosby, Cisneros-Zevallos, & Byrne, 2006). The measurement of the antioxidant activity of food products and ingredients is a matter of growing interest. In addition to the presumed association with health benefits, antioxidants may inhibit, retard or ameliorate oxidative spoilage (Serrano, Goni, & Saura-Calixto, 2007; Tsoukalas, Katsanidis, Marantidou, & Bloukas, 2011; Zulueta, Esteve, & Frigola, 2009). Determination of the total ⇑ Corresponding author. Tel./fax: +86 451 82190535. E-mail address: [email protected] (Y.-J. Fu). 0308-8146/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2013.05.045
antioxidant capacity cannot be performed accurately by any single method due to the diversity of phytochemicals present and the associated chemical moiety diversity (Chu, Chang, & Hsu, 2000). Consequently, at least two different methods should be used to evaluate antioxidant potential capacity. Pyrola, a perennial evergreen herbaceous plant, is mainly distributed in the northern hemisphere in temperate and cold temperate regions around the world. Pyrolae (Chinese name: Lu xian cao, Lu han cao, or Lu ti cao), the whole herb of Pyrola calliantha H. Andr., Passiflora incarnata Fisch., Pyrola decorate H. Andr., or other Pyrola plants (Pyrolaceae), has been used in tonics, sedatives, analgesics against rheumatoid arthritis, and hemostatics (Klyomi et al., 1992). Nowadays, because of its function of slowing down ageing and boosting immunity, it was used as a kind of tea called Lu shou cha for daily drinking in China (Sun et al., 2011). Pyrola is widespread as an edible plant for food industry in landscape area (National Commission of Chinese Pharmacopoeia, 2005). The water extract of the plant was reported to inhibit the growth of many kinds of human pathogenic bacilli in vitro, it can be used in refreshing foods (Dictionary of Chinese Traditional Medicine, 1986). P. incarnata is mainly distributed in the northeast of China. Its’ main constituents are chimaphilin, arbutin, epicatechin, catechin, 200 -O-galloylhyperin, hyperin quercetin, pyrolatin and other naphthoquinones (Kaqawa et al., 1992; Kosuge et al., 1985; Li et al., 2008; Yazaki, Shida, & Okuda, 1989). The main distribution sites of P. incarnata in the northeast of China were Shengshan, Jiagedaqi, Tahe, Wuying, Xinlin, Wudalianchi and Tulihe. It has been well
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known that the content varies in its’ active ingredient, and is affected by environmental, cultural, and post-harvesting conditions. So it is important to evaluate the quality of P. incarnata from different sites in the northeast of China. This variation may aid harvest and the development of P. incarnata as a potential antioxidant resource. Contents of active compounds in P. incarnata samples collected from the above 8 different sites was analysed in this study. The total phenolic contents, total flavonoid contents and the antioxidant activities including tests of scavenging activity, reducing power and b-carotene-linoleic acid bleaching were also determined. The principal component analysis (PCA) was conducted to detect cluster formation and establish relationships among different site samples, active compounds and antioxidant properties. 2. Materials and methods 2.1. Chemicals 1,1-diphenyl-2-picrylhydrazyl (DPPH), b-carotene, linoleic acid, Tween 40, Folin–Ciocalteu reagent, standards of gallic acid, rutin, arbutin, epicatechin, catechin, hyperin, 200 -O-galloylhyperin, quercitrin, pyrolatin, chimaphilin, butylated hydroxytoluene (BHT) and ascorbic acid (Vc) were all purchased from Delta (Wuhu, China). A total antioxidant capacity assay kit (ABTS assay) obtained from Beyotime Institute of Biotechnology (China). Methanol of chromatographic grade was purchased from J & K Chemical Ltd. (China). Formic acid was chromatographic grade (Dima Technology INC., USA). Ethanol was of analytical grade and purchased from Tianjin Chemical Reagents Co. (Tianjing, China). Deionised water was purified by a Milli-Q Water Purification system (Millipore, MA, USA). For standard solutions, appropriate amounts of reference compounds were dissolved in methanol to yield the stock solutions at a concentration of 1 mg/mL, respectively. All solutions prepared for HPLC were filtered through 0.45 lm nylon membranes before used. 2.2. Plant material P. incarnata Fisch. samples were collected in summer from the northeast of China from eight sites (Fig. 1): Tahe forest farm, Xinlin
forest farm, Tulihe forest farm, Jiagedaqi forest farm, Shengshan forest farm, Dayangshu forest farm, Wudalianchi forest farm and Wuying forest farm. The main climate data were shown in Table S1. The samples were identified by Professor Shaoquan Nie from the Key Laboratory of Forest Plant Ecology, Ministry of Education, Northeast Forestry University, PR China. Voucher specimens were deposited in the herbarium of this Key Laboratory. The leaves were naturally dried. After that, the dried leaves were pulverised, and then stored in a dry place at room temperature until used. 2.3. Sample extraction Each samples (10 g) were extracted three times under sonication in an ultrasonic bath (Kunshan Ultrasonic Instrument, Jiangsu, China) with 80% (v/v) aqueous ethanol (1.5 L) at 40 °C for 40 min. The extract liquor was combined three times and concentrated by a rotary evaporator (RE-52AA, Shanghai Huxi Instrument Co., China) to obtain the crude extract. 2.4. Determination of total phenolic and flavonoid contents 2.4.1. The amounts of total phenolics Total phenolic contents of each sample were determined by using the Folin–Ciocalteu method according to Wu et al. (2010) with slight modifications. Briefly, 40 lL of blank and each extract/fractions were mixed with 1.8 mL of Folin–Ciocalteu reagent (10-fold diluted with deionised water). The mixture was allowed to stand for 5 min at room temperature before it was neutralised with the addition of 1.2 mL of 7.5% sodium carbonate solution. After 1.5 h of reaction at ambient temperature and in darkness, the absorbance at 765 nm was measured using spectrophotometer (UNICO, Shanghai, China) and the phenolic contents were calculated using gallic acid as a standard. The total phenolic contents were expressed in milligramme gallic acid equivalents (mg GAE/g extract). All the measurements were taken in triplicate. 2.4.2. The amounts of total flavonoids The flavonoid contents were determined using an aluminium chloride method with rutin as a reference compound. This method is based on the formation of a flavonoid-aluminium complex with an absorptivity maximum at 410 nm (Chang, Yang, Wen, & Chern, 2002). About 0.5 mL of each extract/fractions in 50% ethanol (4 mg/ mL) was added to a 10 mL volumetric flask containing 3 mL of
Fig. 1. Different collection area of pyrola [P. incarnata] in the northeast of China.
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0.1 M potassium acetate (CH3COOK) in deionised water and 2 mL of 0.1 M aluminium chloride hexahydrate (AlCl3). After incubation at room temperature for 20 min, then immediately metered volume to 10 mL with 50% ethanol, the reaction mixture absorbance was measured at 410 nm. Blank sample was used 500 lL of 50% ethanol instead of 500 lL of samples. Three replicates were made for each test sample. The total flavonoid content was expressed as rutin equivalents, in mg/g extract. 2.5. DPPH radical scavenging activity assay The free radical-scavenging activities of samples were measured in terms of hydrogen donating or radical-scavenging ability using the stable radical DPPH (Wu et al., 2009). The different concentrations of the samples in 50% ethanol (100 lL) were mixed with 50% ethanol (1.4 mL) and then added to 0.004% DPPH (1 mL, Sigma–Aldrich) in ethanol. The mixture was shaken vigorously and then immediately incubated in darkness. After 70 min, the reaction reached a plateau. The decreasing of the DPPH solution absorbance was determined in a UV–Vis spectrophotometer (UNICO, Shanghai, China) to monitor absorbance at 517 nm. Ascorbic acid (Sigma–Aldrich), a stable antioxidant, was used as a positive reference. The DPPH radical-scavenging activity in percentage of sample was calculated as follows: DPPH scavenging activity (%) = (1 A517 sample/A517 DPPH solution) 100. 2.6. ABTS radical scavenging activity assay The total antioxidant capacity was evaluated using the ABTS method (Beyotime Institute of Biotechnology, China). ABTS radical cation (ABTS+) solution was produced by reacting ABTS stock solution with 2.45 mmol/L potassium persulfate in the dark at room temperature for 12–16 h before use, and then the resulting ABTS+ solution was diluted with 80% ethanol to adjust the absorbance to 0.70 ± 0.05 at 734 nm. After addition of 10 lL of sample or trolox standard to 200 lL of diluted ABTS+ solution, absorbance at 734 nm was measured after 2–6 min in the dark at room temperature. Trolox, a water-soluble analogue of vitamin E, is used as the reference standard to prepare a calibration curve for a concentration range of 0.15–1.5 mM. Results were expressed as mmol/g Trolox equivalent antioxidant capacity (TEAC). 2.7. The reducing power The reducing power was measured according to the method of Wu et al. (2010) with some modification. An aliquot of each sample (0.5 mL), with different concentrations, was mixed with 0.5 mL of phosphate buffer (0.2 M, pH 6.6) and 0.5 mL of 1% potassium ferricyanide [K3Fe(CN)6]. The reaction mixture was incubated at 50 °C for 20 min. After incubation, 0.5 mL of 10% trichloroacetic acid (TCA) was added, followed by centrifugation at 650 xg for 10 min. The supernatant (0.5 mL) was mixed with 0.5 mL of distilled water and 0.1 mL of 0.1% ferric chloride (FeCl3). The absorbance of all sample solutions was measured at 700 nm. An increased absorbance indicated increased reducing power. BHT was used as the positive control. 2.8. b-carotene-linoleic acid bleaching assay The lipid antioxidant activities of the samples were determined using b-carotene-linoleic acid bleaching assay (Wu et al., 2010). A solution of b-carotene was prepared by dissolving b-carotene (10 mg) in chloroform (10 mL). The b-carotene-chloroform solution (0.2 mL) was pipetted into a round-bottomed flask containing linoleic acid (20 mg) and Tween 40 emulsifier (200 mg). Chloroform was removed using a rotary evaporator at 40 °C for 5 min,
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and distilled water (50 mL) was added to the flask slowly with vigorous agitation to form an emulsion. Aliquots (5 mL) of this emulsion were added into different test tubes containing different concentrations of sample solutions (0.2 mL), and the absorbance was immediately measured at 470 nm against a blank, consisting of an emulsion without b-carotene. The tubes were incubated in a water bath at 50 °C, and the oxidation of the emulsion absorbance was measured at 470 nm over a 60 min period by a spectrophotometer. Control samples contained 200 lL of water instead. BHT, a stable antioxidant, was used as a synthetic reference. The antioxidant activity was expressed as inhibition percentage with reference to the control after a 60 min incubation using the following equation: AA = 100(DRC – DRS)/DRC, where AA = antioxidant activity, DRC = degradation rate of the control = [ln(a/b)/60], DRS = degradation rate in presence of the sample = [ln(a/b)/60], a = absorbance at time 0, and b = absorbance at 60 min.
3. Results and discussion 3.1. Total phenolic and flavonoid contents Phenolic compounds are considered as a major group of compounds that contributed to the antioxidant activity. In order to understand the relationship between antioxidant activity and total phenolic content (TPC), the TPC of P. incarnata samples from 8 different sites in the northeast of China were determined using the Folin–Ciocalteu phenol reagent. The results are expressed in gallic acid equivalents (GAE) and presented in Table 1. The TPC of Tahe and Tulihe were 181.48 ± 3.71 GAE mg/g and 175.09 ± 2.28 GAE mg/g, which were much higher than those of other samples. The lowest TPC was in Shengshan which was only 39.66 ± 1.37 GAE mg/g. The TPC is arranged as following sequence: Tahe > Tulihe > Wudalianchi > Xinlin > Wuying > Dayangshu > Jiagedaqi > Shengshan. Flavonoids are the most commonly and widely distributed group of plant phenolic compounds, which are usually very effective antioxidants. The total flavonoid contents (TFC) of P. incarnata Fisch. from eight different sites in the northeast of China were evaluated by aluminium chloride spectrophotometer. Using rutin as a standard (y = 2.5881x0.0051; r2 = 0.9997). The data (Table 1) show that the highest and lowest flavonoid contents were observed from samples of Tahe and Shengshan, 22.11 ± 1.25 and 2.47 ± 0.34 RE mg/g, respectively. TFC of the Jiagedaqi sample was the second lowest which was only higher than that of Shengshan. TFC is arranged as following sequence: Tahe > Tulihe > Wudalianchi > Xinlin > Wuying > Dayangshu > Jiagedaqi > Shengshan, varied from 2.47 ± 0.34 to 22.11 ± 1.25 RE mg/g. These results were very similar to the results of TPC. TPC and TFC of Tahe were higher than those of other sites. Tahe is located at a high latitude site (Fig. 1) that has the lowest annual average temperature and annual average precipitation among the eight sites. Some literature reports indicate that low temperature stress and stress from water shortage may enhance secondary metabolite content and antioxidant activity (Ill et al., 2006; Zhu, Liang, Han, & Wang, 2009). Low temperature stress may cause damage to cells by inducing reactive oxygen species (ROS) by disrupting the scavenging systems that quench oxygen. And the synthesis of certain phenolic compounds can occur in response to the low temperature stress (Pennycooke, Cox, & Stushnoff, 2005). Water shortage stress may decrease the consumed content of NADPH + H+ within the Calvin cycle. The excess NADPH + H+ is used for the synthesis of secondary metabolites which result in the increase of TPC (Selmar & Kleinwächter, 2013). Thus, the highest TPC and TFC of Tahe may be due to the low annual average temperature and low annual average precipitation. A similar
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Table 1 Total phenolics, flavonoids and antioxidant activity (DPPH, ABTS, reducing power and anti-b-carotene bleaching assay) of different pyrola samples from Northeast China. Sample
Total phenolics (GAE mg/g)
Total flavonoids (RE mg/g)
DPPH IC50a (mg/mL)
ABTSb (mmol/g Trolox)
Reducing power IC50a (mg/mL)
Anti-b-carotene bleaching IC50a (mg/mL)
Shengshan Jiagedaqi Tahe Wuying Xinlin Wudalianchi Tulihe Dayangshu Vc BHT
39.66 ± 1.37 77.76 ± 2.11 181.48 ± 3.71 100.67 ± 6.88 125.52 ± 3.00 145.99 ± 2.44 175.09 ± 2.28 96.47 ± 3.17 – –
2.47 ± 0.34 6.66 ± 0.50 22.11 ± 1.25 10.77 ± 0.83 13.69 ± 0.45 15.46 ± 0.61 19.23 ± 0.65 11.91 ± 1.62 – –
0.282 ± 0.019 0.187 ± 0.010 0.106 ± 0.006 0.160 ± 0.082 0.134 ± 0.010 0.124 ± 0.007 0.112 ± 0.004 0.148 ± 0.015 0.076 ± 0.004 ND
0.254 ± 0.013 0.474 ± 0.020 0.611 ± 0.016 0.521 ± 0.022 0.564 ± 0.043 0.584 ± 0.012 0.643 ± 0.079 0.532 ± 0.017 1.057 ± 0.095 ND
0.318 ± 0.020 0.161 ± 0.018 0.083 ± 0.014 0.162 ± 0.013 0.127 ± 0.008 0.138 ± 0.007 0.102 ± 0.011 0.123 ± 0.010 ND 0.141 ± 0.018
0.089 ± 0.004 0.097 ± 0.004 0.038 ± 0.005 0.042 ± 0.003 0.032 ± 0.004 0.055 ± 0.007 0.061 ± 0.004 0.067 ± 0.004 ND 0.017 ± 0.001
Each value is expressed as the mean ± standard deviation (n = 3). ND, not determined. a IC50 value: the concentration at which the antioxidant activity inhibition ratio reached 50%; 1,1-diphenyl-2-picrylhydrazyl (DPPH) was scavenged by 50%; the absorbance was 0.5 for reducing power; and b-carotene bleaching was inhibition ratio reached 50%. b ABTS were expressed as mmol/g Trolox equivalent antioxidant capacity (TEAC).
Fig. 2. DPPH radical-scavenging activities (A), ABTS radical-scavenging activities (B), reducing power (C), anti-b-carotene bleaching (D) of different pyrola samples in the northeast of China. Each value is expressed as mean ± standard deviation (n = 3).
phenomenon may occur at Tulihe which also has lower annual average temperatures and lower annual average precipitation. 3.2. Antioxidant activity 3.2.1. DPPH radical scavenging activity assay The relatively stable organic radical DPPH has been widely used for the determination of antioxidant activity of pure antioxidant compounds as well as different plant extracts. For evaluation of antioxidant activity of P. incarnata different samples were measured and compared with their DPPH radical scavenging activities. Fig. 2A illustrates the DPPH radical scavenging activities of the different P. incarnata samples. Among the results of DPPH scavenging activities, the Tahe sample had the highest DPPH radical scavenger activity followed by Tulihe, Wudalianchi, Xinlin, Dayangshu,
Wuying, Jiagedaqi and Shengshan. In order to quantify the antioxidant activity further, the IC50 was calculated (Table 1). The lowest IC50 of Tahe was 0.106 ± 0.006 mg/mL reflecting the highest DPPH radical scavenging activity among all samples. The IC50 values of Tulihe, Wudalianchi, Xinlin and Dayangshu were also high, which were only slightly lower than the Tahe samples. From Fig. 1, it can also be seen that when the concentration of samples was 0.25 mg/mL, the DPPH radical scavenging activities of Tahe and Tulihe were more than 85% which were very close to that of Vc indicating that samples from Tahe and Tulihe had the most potent antioxidant activity. 3.2.2. ABTS radical scavenging activity assay The ABTS radical scavenging activity assay is applied to measure the total antioxidant capacity of samples. The results of ABTS
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D.-Y. Zhang et al. / Food Chemistry 141 (2013) 2213–2219 Table 2 The content of the active compounds in different pyrola samples. Sample
Arbutin (mg/g)
Epicatechin (mg/g)
Catechin (mg/g)
Hyperin (mg/g)
20 0 -O-galloyl-hyperin (mg/g)
Pyrolatin (mg/g)
Quercetin (mg/g)
Chimaphilin (mg/g)
Shengshan Jiagedaqi Tahe Wuying Xinlin Wudalianchi Tulihe Dayangshu
0.648 ± 0.047 1.975 ± 0.140 1.695 ± 0.235 2.300 ± 0.132 2.655 ± 0.199 1.875 ± 0.141 2.605 ± 0.178 1.865 ± 0.174
0.108 ± 0.008 0.466 ± 0.038 2.310 ± 0.174 1.053 ± 0.074 1.127 ± 0.014 1.040 ± 0.120 1.810 ± 0.123 1.066 ± 0.089
– 0.120 ± 0.026 0.176 ± 0.010 0.083 ± 0.007 0.081 ± 0.010 0.035 ± 0.003 0.048 ± 0.002 0.050 ± 0.003
0.640 ± 0.033 0.838 ± 0.038 2.742 ± 0.153 1.719 ± 0.085 1.756 ± 0.425 1.771 ± 0.666 2.304 ± 0.228 1.554 ± 0.088
2.300 ± 0.257 3.656 ± 0.236 8.839 ± 0.226 6.331 ± 0.150 6.568 ± 0.197 5.720 ± 0.263 7.423 ± 0.621 6.612 ± 0.335
– – 0.318 ± 0.015 0.124 ± 0.007 – 0.179 ± 0.001 0.251 ± 0.001 –
0.032 ± 0.002 – 0.046 ± 0.002 0.039 ± 0.002 0.047 ± 0.003 0.050 ± 0.003 – –
0.084 ± 0.003 0.172 ± 0.001 0.081 ± 0.008 0.468 ± 0.002 0.416 ± 0.042 0.116 ± 0.001 0.232 ± 0.006 0.405 ± 0.025
radical scavenging activity assay are expressed as trolox equivalent antioxidant capacity (TEAC). In Fig 2B, the highest ABTS+ scavenging activity was found for Tulihe which was differed from the results of the DPPH radical scavenging activity assay. But compared with Tulihe, the TEAC of Tahe and Wudalianchi were not much lower. The TEAC of Shengshan was 0.254 ± 0.013 mmol/g which was still the lowest among the eight samples. The ABTS radical scavenging activity values decreased in the order of Tulihe > Tahe > Wudalianchi > Xinlin > Dayangshu > Wuying > Jiagedaqi > Shengshan. Compared with their DPPH free radical scavenging activities, the trend for ABTS radical scavenging activity did not vary markedly. The TEAC of Vc was 1.057 ± 0.095 (Table 1) which was much higher than that of the P. incarnata samples. Thus, there is a wide gap between Vc and P. incarnata extracts for ABTS radical scavenging activity.
3.2.3. The reducing power It has been reported that reducing power is associated with antioxidant activity and may serve as a significant reflection of the antioxidant activity (Oktay, Gülçin, & Küfreviog˘lu, 2003). Compounds with reducing power indicate that they are electron donors, and can reduce the oxidised intermediates of lipid peroxidation processes, so that they can act as primary and secondary antioxidants (Yen & Chen, 1995). As shown in Fig. 2C, various concentrations of each sample were used for this assay and all of the samples exhibited reducing power. The samples of Tahe, Tulihe, Dayangshu, Xinlin and Wudalianchi revealed good reducing power, with the IC50 of 0.083 ± 0.014, 0.102 ± 0.011, 0.123 ± 0.010, 0.127 ± 0.008 and 0.138 ± 0.007 mg/mL, respectively. The data of the positive control (BHT) was 0.141 ± 0.018 mg/mL. This indicated that the Tahe sample had a remarkable antioxidant capacity better than that of BHT. The Dayangshu sample had better reducing power than that of Xinlin and Wudalianchi although its DPPH and ABTS radical scavenging activity were lower than Xinlin and Wudalianchi.
3.2.4. b-carotene-linoleic acid bleaching assay The b-carotene-linoleic acid bleaching inhibition effect of BHT and eight P. incarnata samples are shown in Fig. 2D. According to Fig. 2D, all the test samples inhibited the oxidation of linoleic acid, with increasing concentration. The IC50 of the eight P. incarnata samples are revealed in Table 1. The results obtained by b-carotene-linoleic acid bleaching inhibition test were different from those of the DPPH method and ABTS method. The Xinlin sample had the best b-carotene-linoleic acid bleaching activity which was very different to the results of DPPH and ABTS test. When the concentration of Xinlin was 0.5 mg/mL, the inhibition could reach to 93.14%. The b-carotene-linoleic acid bleaching activities of Tahe and Xinlin were also relative high. The Tulihe sample which had good DPPH and ABTS radical scavenging activity, had lower b-carotene-linoleic acid bleaching than most of the samples.
All the samples possessed b-carotene-linoleic acid bleaching activities (Fig. 2D, Table 1). 3.3. Constituents comparison of P. incarnata from different sites The constituents of the P. incarnata samples were analysed by HPLC and the data of the 8 principal compounds in plant extracts are presented in Table 2. The most abundant phenolics found in the P. incarnata samples were arbutin, epicatechin, hyperin and 200 -O-galloylhyperin. Other phenolic compounds catechin, pyrolatin, quercitrin and chimaphilin were found at lower concentrations. Not all of the active compounds were detected in the P. incarnata samples, as in catechin in Shengshan, pyrolatin in Shengshan, Jiagedaqi, Xinlin and Dayangshu and quercetin in Jiagedaqi, Tulihe and Dayangshu (Table 2). The highest content of arbutin was in Xinlin and Tulihe followed by Wuying, Jiagedaqi, Wudalianchi, Dayangshu, Tahe and Shengshan. The most abundant component was 200 -O-galloylhyperin ranging from 2.300 ± 0.257 to 8.839 ± 0.226 mg/g. Another higher content compound was hyperin ranged from 0.640 ± 0.033 to 2.742 ± 0.153 mg/g. The highest contents of 2’’-O-galloylhyperin and hyperin, found in the Tahe samples were 8.839 ± 0.226 and 2.742 ± 0.153 mg/g, respectively. Meanwhile, the sample in Tulihe also had high amounts of 200 -Ogalloylhyperin and hyperin. Because of the high latitude, the annual average temperature and precipitation in Tahe is lower, and for Tulihe, its geographic location is in Greater Khingan which can also provide a cold environment for the accumulation of phenolic compounds. The annual average precipitation of Tahe and Tulihe are also smaller (Table S1), drought may induce the accumulation of more phenolic compounds. From the results of Table 1, 200 -O-galloylhyperin could be regard as the main active compound based on content, which is higher than other compounds. According to other research, 200 -O-galloylhyperin has anti-inflammatory, cough, blood pressure, and lower cholesterol, the cardiovascular and cerebrovascular protective role (Kaqawa et al., 1992; Lou, Yang, Song, & Wang, 2004). Thus, P. incarnata Fisch. is an important natural plant resource of 200 -O-galloylhyperin. Hyperin is a famous human health antioxidant in the world, it has been commercially extracted from Hypericum perforatum L., which has faced a big question of resource shortage in recent years. Thus, it is important to find a new resource rich in hyperin. The present result showed that P. incarnata contains abundant hyperin, and could be a potential resource for hyperin. Additionally, the components content of Tahe samples is obviously higher than those of other sites. Thus the Tahe site with low annual average temperature and low annual average precipitation suggests this cool climate site is suitable for the production of antioxidant rich P. incarnata. 3.4. Principal component analysis Principal component analysis (PCA) was performed to understand the interrelationships among the measured antioxidant
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Fig. 3. Principal component analysis loading plot of TPC, TFC, antioxidant activity and active compounds from of different pyrola samples in the northeast of China (A), Principal component analysis score plot of different pyrola samples in the northeast of China (B). RP, reducing power; AC, anti-b-carotene bleaching assay; GH, 200 -O-galloylhyperin.
activity, TPC, TFC and active compounds content. The results of PCA are shown in Fig. 3A. Two principal components, explaining the 80.4% of the total data variance, have been chosen on the basis of their eigenvalues (>1). PC 1 = 0.955 TPC + 0.972 TFC – 0.961 DPPH + 0.947 ABTS – 0.922 RP – 0.770 AC + 0.667 arbutin + 0.951 epicatechin + 0.557 catechin + 0.967 hyperin + 0.966 GH + 0.763 pyrolatin + 0.193 quercetin + 0.149 chimaphilin. PC 2 = 0.195 TPC + 0.175 TFC + 0.153 DPPH – 0.195 ABTS + 0.232 RP + 0.095 AC – 0.649 arbutin + 0.190 epicatechin + 0.130 catechin + 0.168 hyperin – 0.081 GH + 0.547 pyrolatin + 0.398 quercetin – 0.897 chimaphilin.
The first principal component (PC1) correlated well with TPC, TFC, DPPH, ABTS, reducing power, epicatechin, hyperin and 200 -Ogalloylhyperinwith the loadings 0.955, 0.972, 0.961, 0.947, 0.922, 0.951, 0.967 and 0.966, respectively. The second principal component (PC 2) was related to chimaphilin with loadings of 0.897. There were good correlations between TPC, TFC, DPPH and ABTS and these were consistent with the results in Fig 3A. Moreover, DPPH radical scavenging activity, b-carotene-linoleic acid bleaching assay, and reducing power were found to be similarly loaded on PC 1, which indicated the three properties were closely related to antioxidant activity. TPC and TFC also had high loading on PC1, which suggested phenolic compounds were good
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antioxidants. For PC 2, only chimaphilin get high loadings, while the antioxidant activity (DPPH radical scavenging activity, ABTS radical scavenging activity, b-carotene-linoleic acid bleaching assay, and reducing power) had low loading, which indicated that chimaphilin might not have good antioxidant activity. The content of 200 -O-galloylhyperin which correlated well with DPPH radical scavenging activity, ABTS radical scavenging activity and b-carotene-linoleic acid bleaching assay, was higher than other compounds. Thus, 200 -O-galloylhyperin can be regard as the main antioxidant component in P. incarnata Fisch. On the other hand, epicatechin, catechin and hyperin also has good antioxidant activity from some other reports (Rusak, Komes, Likic, Horzˇic´, & Kovacˇ, 2008; Xing et al., 2011) and this is also similar to our correlation analysis results. PCA can also provide an overview of the similarities and differences among the 8 P. incarnata samples from different areas. The Tahe sample found in the upper right part of Fig. 3B, which was positively correlated with PC1 and PC2. This sample can be best described as having high antioxidant activity and a high content of total phenolics and flavonoids. Close relationships were observed among Wuying, Dayangshu and Xinlin which indicated that they have similar antioxidant activity and contents of total phenolic and flavonoid. While the sample of shengshan had the largest negative score of PC 1, it indicated that shengshan exhibited the lowest antioxidant activity and contents of total phenolic and flavonoid.
4. Conclusions Pyrola [P. incarnata], grown in Northeast China, is a potentially rich source of desirable health attributes. The Tahe sample has high total phenolic and flavonoid contents and good antioxidant activity. Its’ IC50 of reducing power test is 0.083 ± 0.014 mg/mL which is better than that of BHT (0.141 ± 0.018 mg/mL) and the IC50 (0.106 ± 0.006 mg/mL) of DPPH test is also close to that of Vc (0.076 ± 0.004 mg/mL). These results indicate that the pyrola of Tahe possesses remarkable antioxidant activity. Moreover, eight compounds of different P. incarnata samples were quantified. 200 -O-galloylhyperin and hyperin were the most abundant components, and P. incarnata can be regarded as a potential natural plant resource of these two compounds. The Tahe sample also has a high content of active compounds including 200 -O-galloylhyperin, hyperin, arbutin and epicatechin. PCA indicates that P. incarnata from Tahe has the best quality, it could be a valuable antioxidant natural source in the northeast of China. The present study supports meaningful information for the collection and application of the P. incarnata resource in food production.
Acknowledgements The authors gratefully acknowledge the financial supports by Program for Agricultural Science and Technology Achievements Transformation Fund Program (2012GB23600641), Special Fund of Forestry Industrial Research for Public Welfare of China (201004040), Importation of International Advanced Forestry Science and Technology, National Forestry Bureau (2012-4-06) and Heilongjiang Province Science Foundation for Excellent Youths (JC200704).
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.foodchem.2013. 05.045.
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