Anthocyanins composition and antioxidant activity of two major wild Nitraria tangutorun Bobr. variations from Qinghai–Tibet Plateau

Food Research International 44 (2011) 2041–2046

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Anthocyanins composition and antioxidant activity of two major wild Nitraria tangutorun Bobr. variations from Qinghai–Tibet Plateau Jie Zheng a,c, Hui Li b,c, ChenXu Ding a, YouRui Suo a,⁎, LiangSheng Wang b, HongLun Wang a a b c

Northwest Institute of Plateau Biology, Chinese Academy of Sciences, Xining 810001, China Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China Graduate University, Chinese Academy of Sciences, Beijing 100049, China

a r t i c l e

i n f o

Article history: Received 19 April 2010 Accepted 12 July 2010 Keywords: Nitraria tangutorun Bobr. Anthocyanins Total polyphenol content Antioxidant activity HPLC–MS

a b s t r a c t Nitraria tangutorun Bobr., a unique kind of fruit, widely spreads in the Qinghai–Tibet Plateau. In the present study, nine anthocyanins were identified in two variations (purple fruit and red fruit) of N. tangutorun by HPLC/DAD–ESI/MS. Cyanidin-3-O-(trans-p-coumaroyl)-diglucoside (215.76 ± 22.91 mg of Mv3G5G equivalent per 100 g of fresh weight) and pelargonidin-3-O-(p-coumaroyl)-diglucoside (5.13 ± 0.35 mg of Mv3G5G equivalent per 100 g of fresh weight) were the main anthocyanins in the purple and red fruits respectively. In addition, most of the anthocyanins were acylated by coumaric acid, and the rare anthocyanins that naturally presented a coumaric acid in both cis and trans configurations have been detected. Furthermore, the extract of the two variations showed significantly different antioxidant activity (p b 0.01) according to DPPH, ABTS and FRAP assay. Purple fruit possessed higher antioxidant activity than red fruit. There were significant correlations between antioxidant activity and both the total polyphenol content and anthocyanins content. This work is valuable for elucidation of anthocyanins composition in N. tangutorun and for further utilization as a functional food and medicine material. © 2010 Elsevier Ltd. All rights reserved.

1. Introduction Nitraria tangutorun Bobr. is a unique fruit that is widely distributed in salinized desert of the Qinghai–Tibet Plateau. Its special physiological characteristics of drought-resistance and salt-resistance make it an ideal plant for preventing soil desertification and alleviating the degree of soil salinity–alkalinity (Zhao, Fan, & Ungar, 2002), which is very important for the ecosystem and agriculture in the remote area (Li, Zhang, & Zhang, 2006). Beside the remarkable ecological values, its fruits have been used as a nutritional food and traditional herb for the treatment of hypertension, abnormal menstruation and indigestion among folks in the Northwest of China, and adding certain amount of N. tangutorun fruits to the daily diet of confined women could help the milk-secretion during lactation (Li et al., 2006). But as a highly evolutionary species in the Nitraria genera, its intra-species mutation is intense. Especially the variability of its fruit is more

Abbreviations: HPLC, high performance liquid chromatography; DAD, diode array detector; ESI, electrospray ionization; MS, mass spectrometry; DPPH, 1,1-diphenyl-2picrylhydrazyl; TPTZ, 2,4,6-tripyridyl-S-triazine; ABTS, 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid); FRAP, ferric reducing antioxidant power; Mv3G5G, malvidin-3,5-di-Oglucoside chloride; GA, gallic acid; FW, fresh weight; TPC, total polyphenol content; SPE, solidphase extraction cartridge. ⁎ Corresponding author. Northwest Institute of Plateau Biology, Chinese Academy of Sciences, Xiguan Avenue 59 #, Xining, 810001, China. Tel./fax: +86 0971 6143857. E-mail address: [email protected] (Y. Suo). 0963-9969/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodres.2010.07.008

intense than that of other nutritional organs. Red and purple fruits of N. tangutorun are the variations mainly encountered in the wild (Gao, Cui, Liu, & Wang, 1998). N. tangutorun presents great ecological and pharmaceutical effects, but to our best knowledge, so far no researches about the chemical composition of its variations have been conducted. Nowadays all the variations have been used as a food or medicine material without distinction. This has largely restricted the further research and development of N. tangutorun. Li, Zhang, and Wang (2005) have reported that N. tangutorun fruit had abundant anthocyanins. Its pleasing color has aroused scholars to investigate it, but most of the researches were focused on the extraction method and total anthocyanins content measurement. To this day, no comprehensive study has been conducted to explore the anthocyanins composition of N. tangutorun variations. Anthocyanins, a major kind of natural pigment, diffusely exist in colorful fruits, flowers and leaves. They are responsible for the brilliant colors of the plant (red, blue or purple). The anthocyanin is composed of an aglycone and sugar moieties, in some cases the sugar moieties are acylated by organic acid. Up to now, there are 23 aglycones that have been reported, six of which are the most common in vascular plants, including pelargonidin, cyanidin, delphinidin, peonidin, petunidin and malvidin (Castaneda-Ovando, Pacheco-Hernández, Páez-Hernández, Rodríguez, & Galán-Vidal, 2009). The structure of anthocyanins is highly variable, due to the number of hydroxyl, the nature and the number of sugar moieties, the aliphatic or aromatic acids linked to sugar in the molecule and the position of these bonds (Kong, Chia, Goh, Chia, &

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Brouillard, 2003). The great structure diversity endows anthocyanins with a wide range of biological and physiological effects such as antioxidant activity, anticardiovascular disease and anticarcinogenic effects (Ichikawa et al., 2001; Lamy et al., 2006; Rahman, Ichiyanagi, Komiyama, Sato, & Konishi, 2008; Wang, Cao, & Prior, 1997). The likely mechanism is postulated as that anthocyanins act as potent antioxidants by donating hydrogen atoms to highly reactive free radicals, breaking the free radical chain reaction (Rice-Evans, & Miller, 1996). Moreover, it is well known that degenerative and pathological processes such as atherosclerosis, aging, and cancer in the human body are related to oxidative damage (Shi, Gong, Liu, Wu, & Zhang, 2009). Anthocyanins as a major component in N. tangutorun, and identifying its composition would help us to understand more about the unique species and to select the improved breed. As HPLC–MS technique has developed rapidly during the last century, anthocyanins identification has become possible within a short time according to UV/Vis and MSn data. For antioxidant activity evaluation, the 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) and radical monocation of 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and ferric reducing antioxidant power (FRAP) are widely employed to evaluate antioxidant activity of extracts or single compounds (Brand-Williams, Cuvelier, & Berset, 1995; Fang et al., 2009). For the further utilization of N. tangutorun, the objective of this study is to establish a reliable method to investigate the anthocyanins' composition and antioxidant activity of two major N. tangutorun variations. 2. Material and methods

activity assay, 1 mL of filtrate was evaporated at 30 °C, and the residue was dissolved in 1 mL of water and then purified by solid-phase extraction cartridge (SPE), C18 Supelclean ENVI-18 cartridge (Supelco park, Bellefonte, PA, 500 mg, 3 mL), which had been previously activated with methanol and water. The cartridge was successively rinsed with water (to remove sugars, formic acid, and other interfering substances) and methanol (to elute the polyphenolic fraction). The methanolic eluate was concentrated at 30 °C, and the residue was redissolved in 1 mL of methanol for Folin–Ciocalteu and antioxidant activity assay. 2.4. HPLC/DAD analysis The samples were analyzed by Dionex HPLC system (Sunnyvale, CA, USA), equipped with P680 HPLC pump, UltiMate 3000 autosampler, TCC-100 thermostated column compartment and Dionex PDA100 photodiode array detector. The analytical column was C18 column of ODS 80Ts QA (150 mm × 4.6 mm, 5 μm i.d.,Tosoh, Tokyo, Japan) protected with a C18 guard cartridge (Shanghai ANPEL Scientific Instrument, Shanghai, China). An aliquot of 10 μL solution was injected. Chromatograms were obtained at 525 nm for anthocyanins, and the UV/ Vis spectrum was recorded from 200 to 800 nm. Gradient program for anthocyanins analysis was applied. The eluents were: A, 10% aqueous formic acid with 0.1% TFA; B, 15% methanol in acetonitrile. The applied gradient program was: 0 to 20 min, linear gradient from 3% to 5% B; 20 to 40 min, 5% B isocratic; 40 to 55 min, linear gradient from 5% to 12% B; 55 to 70 min, linear gradient from 12% to 22% B; 70 to 100 min, linear gradient 22%–3% B. The flow rate was 0.8 mL min−1, and the temperature was 35 °C.

2.1. Chemicals 2.5. HPLC–ESI/MS analysis Malvidin-3,5-di-O-glucoside chloride (Mv3G5G) was purchased from Extrasynthese (Genay, France). Gallic acid (GA) was purchased from Must Bio-Technological Co., Ltd (Chengdu, China). Trifluoroacetic acid was from Merck (Hohenbrunn, Germany), DPPH, ABTS, 2,4,6tripyridyl-S-triazine (TPTZ) and Folin–Ciocalteu's phenol reagent were purchased from Sigma-Aldrich (St. Louis, MO). Methanol and acetonitrile used for HPLC/DAD–ESI/MS analysis were of chromatographic grade and purchased from Alltech Scientific (Beijing, China). Methanol and formic acid for anthocyanins extraction were of analytical grade and purchased from Beijing Chemical Works (Beijing, China). HPLC grade water was obtained from Milli-Q System (Millipore, Billerica, MA, USA). 2.2. Plant material Two different fresh fruits (purple and red) of N. tangutorun were randomly sampled from Delingha (Latitude. 37°13′N, Longitude. 97°14′E, Altitude. 2980 m). The fruits were hand-picked in August, 2009, then stored in heat preservation box with efficient ice bag right after collection. All samples were preserved at −20 °C in the laboratory for later analysis. 2.3. Extraction and preparation of the anthocyanins The anthocyanins extraction method was modified from the method used by Zhang et al. (2007) with slight modification. 8 g (purple fruit) and 20 g (red fruit) were extracted triply with 10 mL 2% formic acid in methanol separately. The procedure was conducted in 50 mL conical beaker with its orifice sealed at room temperature in a dark place for 24 h. Then the solution were combined and separated from the solid matrix (fruits remnant, protein and polysaccharide sediment) by filtrating through a sheet of qualitative filter paper (Hangzhou Special Paper Industry, Zhejiang, China). The filtrate was further passed through a 0.22 μm reinforced nylon membrane filter (Shanghai ANPEL, Shanghai, China) for HPLC analysis. For the Folin–Ciocalteu and antioxidant

Mass spectrometry system was an Agilent-1100 HPLC system coupled with UV/Vis detector and ion trap mass detector (Agilent Technologies, Palo Alto, CA, USA). The chromatographic separation condition was described above. The MS conditions were listed as follows: positive ion mode; gas (N2) temperature, 350 °C; flow rate, 8 L min−1; nebulizer pressure, 35 psi; HV voltage, 4 KV; octopole RF amplitude, 150 Vpp; skim 1 voltage, 47.7 V; skim 2 voltage, 6.0 V; capillary exit, 127.3 V; cap exit offset, 79.6 V and scan range, m/z 0–1200. 2.6. Quantitative analysis of anthocyanins Anthocyanins in each samples were semi-quantified by Mv3G5G with the final concentration ranging from 0.01 to 0.80 mg mL−1, and the calibration curve was Y (peak area) = 504.06 × X (Mv3G5G equivalent content) + 0.2275 (r = 0.9999). The content was expressed as milligrams of Mv3G5G equivalent per 100 g of fresh weight (FW). All of the samples were analyzed in triplicate. 2.7. Estimation of the total polyphenol content Total polyphenol content (TPC) in the extract was determined according to the Folin–Ciocalteu Method (Mansouri, Embarek, Kokkalou, & Kefalas, 2005). GA was used as standard compound. The TPC was calculated from the calibration curve Y (absorbance) = 4.3781 × X (GA equivalent content) + 0.0523 (r = 0.9994), and the results were expressed as milligrams of GA equivalents per 100 g of FW. 2.8. DPPH assay The DPPH assay was performed according to the method used by Li et al. (2009) with slight modification, and the method was used widely to evaluate the free radical scavenging activity of plant extraction due to its stable character (Brand-Williams et al., 1995). GA was used as the reference compound. The scavenging percentage

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of DPPH in each sample was calculated according to the formula: absorbance (λ = 515nm) 0 −absorbance (λ = 515nm) t /absorbance (λ = 515nm) 0, where t stands for the time that the reaction reached the plateau stage, and 0 represents the beginning of the reaction. The scavenging ratio was plotted against GA concentration, and the amount of GA equivalents in each sample was calculated by calibration curve Y (scavenging ratio)= 0.3786×X (GA equivalents content)+0.1632 (r=0.9991). All of the samples were analyzed in triplicate. 2.9. ABTS assay Previous literature was taken as a reference (Re et al., 1999). ABTS reagent was produced by reacting 10 mL of 7 mM ABTS solution with 178 μL of 140 mM potassium persulfate aqueous in the dark at room temperature for 13 h before use. The ABTS solution was diluted to appropriate absorbance (0.2–0.8 absorbance). 1 μL sample was added to 799 μL diluted ABTS solution to react in the dark at room temperature for 10 min, and the absorbance at 732 nm was recorded. GA was used as standard compound with the final concentration ranging from 0.25 mM to 2 mM, and the calibration curve was Y (scavenging ratio)=0.4635×X (GA content) +0.0075 (r= 0.9973). Results were expressed as GA equivalent (GA equivalent mg per 100 g of FW). All of the samples were analyzed in triplicate.

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3.2. Anthocyanins' identification The individual anthocyanin was identified mainly by their HPLC retention time, elution order, UV/Vis spectrum, comparison of MS spectra to previously reported data, and guidelines previously introduced by Giusti, Rodriguez-Saona, Griffin, and Wrolstad (1999). Although eight major peaks were visible, mass spectrometry analysis revealed that peak 5 corresponded to two different coeluted anthocyanins. This represents a total of nine anthocyanins in both variations. The coeluted anthocyanins are structurally different but have the same retention time. Coeluted phenomenon is common in anthocyanins investigation (Nicoué, Savard, & Belkacemi, 2007). It is very difficult to check the peak purity with only a DAD detector. But we can examine the peak purity simultaneously with online-MS. In case, if HPLC–MS were unavailable, different column or gradient programs would be an ideal method to distinguish the coelution phenomenon. Since, coelution phenomenon could result in complicated mass spectrometry and

2.10. FRAP assay The FRAP assay was based on the method of Benzie, and Strain (1996). Reagents included 300 mM acetate buffer (pH 3.6); 10 mM TPTZ in 40 mM hydrochloric acid; 20 mM FeCl3·6H2O. The FRAP reagent was prepared by mixing 25 mL acetate buffer, 2.5 mL TPTZ solution and 2.5 mL FeCl3·6H2O solution. 10 μL sample was added into the 790 μL FRAP solution, the reaction was maintained for 20 min when the absorbance reading was constant at 593 nm. GA was introduced as standard compound with the final concentration ranging from 0.25 mM to 2 mM, and the calibration curve was Y (absorbance)= 0.0352 × X (GA content) + 0.1376 (r = 0.9989). The results were expressed as GA equivalent (GA equivalent mg per 100 g of FW). All of the samples were analyzed in triplicate. 2.11. Statistical analysis All samples were measured in triplicate. Statistical analysis was done by Independent-Samples t test. The Pearson correlation coefficient (r) and p-value were calculated to show correlations and their significance by SPSS 16.0. Probability values of p b 0.05 and p b 0.01 were considered statistically significant and extremely significant respectively. 3. Results and discussion 3.1. HPLC analysis The RP-HPLC method provided repeatable and good separation for both variations (Fig. 1). Totally, eight major peaks have been detected in each variation. Based on the chromatograms, the anthocyanins' fingerprints of both variations were similar, whereas the anthocyanins content varied in each variation. The major anthocyanin of the purple fruit was peak 7, and its content was almost 73-fold more than that of the red fruit, in spite that peak 8 was the most abundant anthocyanin in red fruit, which was still lower than purple fruit (almost 5-fold difference). Moreover, anthocyanins have been successfully used as a biochemical marker in taxonomical studies, and with the unique anthocyanins' fingerprints we can possibly distinguish the two N. tangutorun variations from each other (OrtegaRegules, RomeroCascales, LopezRoca, RosGarcia, & GomezPlaza, 2006; Ryan, & Revilla, 2003).

Fig. 1. The detailed chromatograms at 525 nm, (a) the anthocyanins composition chromatogram of purple fruit, and the inserted figure is an enlarged partial chromatogram (40.00–66.00 min); (b) the anthocyanins composition chromatogram of red fruit, and the inserted figure is an enlarged partial chromatogram (35.00–68.00 min). Peaks: 1, cyanidin-3-O-diglucoside; 2, pelargonidin-3-O-diglucoside; 3, delphinidin-3-rutinoside; 4, peonidin-3-O-diglucoside; 5, cyanidin-3-O-(caffeoyl)-diglucoside and delphinidin-3-O(caffeoyl)-diglucoside; 6, cyanidin-3-O-(cis-p-coumaroyl)-diglucoside; 7, cyanidin-3-O(trans-p-coumaroyl)-diglucoside; and 8, pelargonidin-3-O-(p-coumaroyl)-diglucoside.

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unable to exactly quantify the target compound, long time gradient program was applied in the present study to maximally separate the anthocyanins. Some minor peaks other than the eight major peaks have been detected in the study, but due to the low signal-to-noise ratio we cannot identify them yet. The determination of the molecular weights by ESI–MS/MS showed that only four of the most widespread anthocyanins, namely, delphinidin (m/z 303), cyanidin (m/z 287), peonidin (m/z 301) and pelargonidin (m/z 271) were found in N. tangutorun variations. The detailed data of all identified anthocyanins are summarized in Table 1. Peak 1 was the first anthocyanin eluted from C18 column. Taken the retention time (tR = 41.733 min), the MS data (M+ = m/z 611; MS/ MS = m/z 287) and the neutral loss (324 Da) into consideration, we speculated that there were two hexoses attached to the aglycone. Mass spectrum did not detect other fragments except m/z 611 and 271. Probably the two sugars as diglucoside substitution linked to the same position of the aglycone. The cleavage of the glycosidic bond resulted in two sugars departing from the anthocyanin simultaneously. Consequently MS did not detect anthocyanin fragment with one sugar moiety. The MS spectra were in agreement with the anthocyanin reported in red cabbage (Wu, & Prior, 2005). Furthermore, Kong et al. (2003) suggested that the presence of the 3-O-glucoside derivatives of anthocyanins is 2.5 times more frequent than the 3,5-O-diglucosides ones. Lastly, peak 1 was tentatively identified as cyanidin-3-O-diglucoside. The same MS fragmentation pattern was found in peak 2 (M+ = m/z 595; MS/ MS = m/z 271) and peak 4 (M+ = m/z 625; MS/MS = m/z 301). According to the above guidelines, peak 2 and 4 were tentatively identified as pelargonidin-3-O-diglucoside and peonidin-3-O-diglucoside respectively. As a result of the limited information available, the exact structures of these hexoses could not be elucidated yet. MS data (m/z 611/465/303) indicated that peak 3 was a delphinidin derivative with one hexose and pentose respectively. According to the previous research, rutinosides and neohesperidosides were the most common disaccharides encountered in flavanoids (Abad-García, Berrueta, Garmón-Lobato, Gallo, & Vicente, 2009). They are isomers which only differ by the interglycosidic linkage between glucose and rhamnose (rhamnosyl-(α1 → 6)-glucose for rutinose; rhamnosyl-(α1 → 2)-glucose for neohesperidose). Rutinose is more susceptible to lose rhamnose than neohesperidose (Abad-García, Garmón-Lobato, Berrueta, Gallo, & Vicente, 2009). So we can detect the fragment of anthocyanin with one hexose (m/z 465). Combining

the above information, the structure of peak 3 was elucidated as delphinidin-3-O-rutinoside. Peak 5 was a mixture, corresponding to two different anthocyanins. Delphinidin (m/z 303) and cyanidin (m/z 287) were detected simultaneously by MS. The two anthocyanins share the same molecule weight of m/z 773. In addition to the MS information, the retention time (tR = 61.975 min) and previous reports (Wu, Prior, & USDA, 2005; Wu et al., 2005) were taken into consideration. The neutral loss (162 Da) and long retention time indicated that both anthocyanins were acylated by caffeic acid. But due to lack of two dimensional NMR data and relevant reports, the ester bond position was not certain. Lastly, the mixture was identified as cyanidin-3-O-(caffeoyl)-diglucoside and delphinidin-3-O-(caffeoyl)-diglucoside respectively. Peaks 6 and 7 were a pair of isomers. They had the same molecular weight (M+ = m/z 757) and fragment (MS/MS = m/z 287). Nicoué et al. (2007) have reported that acylation of the sugar moieties of the anthocyanins causes a loss of polarity, increasing the retention time. On the basis of the long retention time (tR = 62.925, 66.825 min respectively) and high molecular weight, peaks 6 and 7 should be acylated anthocyanins. Moreover, the UV/Vis spectrum (200– 800 nm) presented a strong absorbance band around 315 nm other than the charactering maximal absorbance band around 525 nm, the characteristic UV/Vis spectrum further confirmed that the anthocyanins were acylated (Slimestad, & Solheim, 2002). According to the MS data (M+ = m/z 757; MS/MS = m/z 287; neutral loss 470 Da = diglucoside 324 Da + p-coumaric acid 146 Da) and UV/Vis information, the anthocyanin was determined as cyaniding derivative with two hexoses and a p-coumaric acid. Additionally, McDougall, Fyffe, Dobson, and Stewart (2007) have reported the diglucoside-coumaroyl substituent would easily depart from anthocyanin aglycone, and very limited MS/MS fragments data would be detected, which was in accordance with the fragmentation patterns encountered in peaks 6 and 7. For the elution order, previous studies reported cis-p-coumaroyl derivatives had higher polarity than its trans configuration, and eluted earlier (Downey, & Rochfort, 2008; George et al., 2001). Eventually, peaks 6 and 7 were identified as cyanidin-3-O-(cis-p-coumaroyl)-diglucoside and cyanidin-3-O-(trans-p-coumaroyl)-diglucoside respectively. Peak 8 had the similar fragmentation pattern (M+ = m/z 741; MS/ MS = m/z 271) with peaks 6 and 7. The only difference was the anthocyanin aglycone. Peak 8 was a pelargonidin derivative with two hexoses and a p-coumaric acid. But with the limited information the actual cis/trans type could not be elucidated yet. Eventually, peak 8 was identified as pelargonidin-3-O-(p-coumaroyl)-diglucoside. 3.3. The anthocyanins content

Table 1 Anthocyanins identified from the methanol extract of N. tangutorun (purple fruit taken as an example). UV λmax M+ MS/MS (nm) (m/z) (m/z)

Peak Category no.

tR (min)

1 2

Cyanidin-3-O-diglucoside Pelargonidin-3-O-diglucoside

611 595

287 271

3

Delphinidin-3-rutinoside

611

4

Peonidin-3-O-diglucoside

625

465/ 303 301

5

Cyanidin-3-O-(caffeoyl)-diglucoside Delphinidine-3-O-(caffeoyl)diglucoside Cyanidin-3-O-(cis-p-coumaroyl)diglucoside

44.942 277.90 53.883 258.77 510.25 58.958 256.04 518.45 61.508 283.37 518.45 61.975

773 773

287 303

65.925 280.64 318.91 521.18 66.825 286.10 313.44 521.18 70.558 280.64 316.17 507.52

757

287

757

287

741

271

6

7

Cyanidin-3-O-(trans-p-coumaroyl)diglucoside

8

Pelargonidin-3-O-(p-coumaroyl)diglucoside

Fig. 2 presents the main anthocyanins (peaks 7 and 8) content of different variations. Clearly, the anthocyanins content significantly varied from each other. Especially, peak 7 (cyanidin-3-O-(trans-p-coumaroyl)diglucoside), its content is largely increased in the purple fruit (almost 73 times higher than that of the red fruit), and reached 215.76 ±22.91 mg Mv3G5G per 100 g of FW. While peak 8 (pelargonidin-3-O-(pcoumaroyl)-diglucoside) content only increased almost 5 times, and up to 24.16 ±6.13 mg Mv3G5G equivalent per 100 g of FW in purple fruits. Cyanidin-3-O-(trans-p-coumaroyl)-diglucoside increased dramatically in purple fruits and possibly caused the color change from red to purple. As for the anthocyanins composition, cyanidin derivatives accounted for almost 80% of the total anthocyanins peak area in purple fruit, which indicated that the anthocyanins accumulation pattern was single for N. tangutorun. The solely accumulation of the cyanidin derivatives in purple fruit of N. tangutorun could be attributed to the high activity of flavonoid 3′-hydroxylase enzyme, which is a critical enzyme to produce dihydromyricetin in the anthocyanins biosynthesis pathway (Holton, & Cornish, 1995). Additionally, we speculated that the rapid increase of acylated anthocyanins was related to the local climate. The fruit was ripe in August when the sunshine time and intensity reached the maximum in

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Table 2 Correlation coefficients of the anthocyanins content, TPC, DPPH, ABTS and FRAP.

Cyanidin-3-O-(trans-p-coumaric acid)-diglucoside Perlagonidin-3-O-(p-coumaric acid)-diglucoside TPC

DPPH

ABTS

FRAP

0.977** 0.911* 0.940**

0.998** 0.961** 0.943**

0.995** 0.952** 0.955**

**Correlation is significant at the 0.01 level (2-tailed). *Correlation is significant at the 0.05 level (2-tailed).

Fig. 2. The major anthocyanins' content in two wild N. tangutorun variations (Mean ± SD, n = 3, bar with ** are significantly different at the 0.01 level (2-tailed)).

the year, the abundant stable acylated anthocyanins could prevent the plant from sunshine harm, which is important for the plant to survive in such an ultraviolet rich area (Holton, & Cornish, 1995; Sadilova, Stintzing, & Carle, 2006). 3.4. Total polyphenol content and antioxidant activity Both of the variations have been taken for TPC quantitative analysis and antioxidant activity evaluation, and the results are displayed in Fig. 3. The results displayed significant differences between variations under Independent-Samples t test (p b 0.01). TPCs for each variation were 164.59 ± 0.88 (red fruit) and 401.61 ± 59.66 (purple fruit) mg GA equivalent per 100 g of FW. The TPC was almost 2.5 times higher in purple fruit than the red fruit. The variations showed extremely significant difference in each antioxidant assay under Independent-Samples t test (p b 0.01). All of the results were expressed as milligram GA equivalent per 100 g of FW. In DPPH assay, the antioxidant activity was 308.37 ± 31.54 mg GA equivalent per 100 g of FW for purple fruit, while 58.16 ± 2.29 mg GA equivalent per 100 g of FW for red fruit. The results indicated that the purple fruit not only obtained more anthocyanins, but also more TPC

than its counterpart. As for ABTS and FRAP assay, the antioxidant activity were 95.78 ± 0.95 (red fruit) and 459.00 ± 25.92 (purple fruit) mg GA equivalents per 100 g of FW (ABTS), and 161.11 ± 5.39 (red fruit) and 886.73 ± 48.42 (purple fruit) mg GA equivalents per 100 g of FW (FRAP). In both assays, the purple fruit exhibited higher antioxidant activity (p b 0.01). These results indicated that the purple fruit could be used as an ideal resource for anthocyanins and polyphenol, and likely possessed more potent curative effect in some illness treatment. 3.5. Correlation analysis Positive correlation coefficients of anthocyanins content, TPC, DPPH, ABTS, and FRAP were observed and listed in Table 2. Results showed that the major anthocyanins content (cyanidin-3-O-(trans-p-coumaroyl)diglucoside and pelargonidin-3-O-(p-coumaroyl)-diglucoside) and TPC correlated extremely high (p b 0.01) with DPPH, ABTS, and FRAP assay, except for the correlation coefficients (r = 0.911⁎) for pelargonidin-3-O(p-coumaroyl)-diglucoside and DPPH. An obvious trend has been found that the variation with larger TPC had the more efficient antioxidant activity. The results were in accordance with previous report (RiceEvans, & Miller, 1996). Both TPC and major anthocyanins correlated significantly with DPPH, ABTS and FRAP, which meant that anthocyanins and the TPC all contributed to the antioxidant activity of N. tangutorun extract. The extract from the purple fruit had the higher antioxidant activity, which might partially be due to its higher anthocyanins and TPC content. Further, the results implied that purple fruit of N. tangutorun might be a potential resource for the development of antioxidant function drinks. 4. Conclusions The present study provided the chemical basis for setting up anthocyanins fingerprints of two major variations of N. tangutorun. In total, nine anthocyanins have been identified by our established method. Cyanidin derivatives were the most abundant anthocyanins, and accounted for almost 80% of the total anthocyanins content. Due to the potential high stability, pleasing color and potent antioxidant activity, it could be considered as natural colorant or beneficial food. Furthermore, we only take a glimpse into N. tangutorun chemical composition in the study. For further understanding of its physiological and pharmacological properties, more works are needed to be carried out. In conclusion, N. tangutorun rich in anthocyanins would be selected for intense use. Acknowledgement

Fig. 3. The antioxidant activity of the methanol extract of two wild N. tangutorun variations (Mean ± SD, n = 3, bar with ** are significantly different at the 0.01 level (2-tailed)).

We thank Dr. ChongHui Li and Jie Zhang (Institute of Botany, Chinese Academy of Sciences) for their help and suggestions in the study, YanJun Xu (China Agricultural University) for his help in HPLC–MS analysis, and Dr. JunYou Shi (Northwest Institute of Plateau Biology, Chinese Academy of Sciences) for his help in material collection. The study was supported by Important Science & Technology Specific Projects of Qinghai Province, China (Grant no. 20099A2-2) and National Natural Science Foundation of China (Grant no. 30873158).

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