Various antioxidant effects were attributed to different components in

Various antioxidant effects were attributed to different components. 1 in the dried blossoms of Citrus aurantium L. var. amara Engl. 2. Chun-Yan Shen,...
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Various Antioxidant Effects Were Attributed to Different Components in the Dried Blossoms of Citrus aurantium L. var. amara Engl ABSTRACT: Citrus aurantium L. var. amara Engl. (CAVA) was traditionally used as an edible and medicinal material in China. Total flavonoids (CAVAF), alkaloids (CAVAA), polysaccharides (CAVAP), coumarins (CAVAC), and neroli (CAVAO) were extracted from CAVA. Hesperidin, naringin, and neohesperidin composed 83.94% of CAVAF, and synephrine represented 50.56% of CAVAA. On the basis of 1,1-diphenyl-2-picrylhydrazyl radical (DPPH•), 2,2′-azinobis(3-ethylbenzothiazoline-6sulfonic acid) diammonium salt radical cation (ABTS• +), hydroxyl radical (•OH), ferric-reducing antioxidant power (FRAP), and reducing power assays, the antioxidant activities of five components were comprehensively and comparatively investigated. CAVAF had a stronger DPPH• scavenging effect and FRAP and reducing power. CAVAP and CAVAA exhibited comparable • OH scavenging effects to vitamin C. CAVAA showed the highest ABTS• + scavenging activity. In conclusion, different constituents varied significantly toward different sources of free radicals and other oxidants. It is obvious that CAVA has various antioxidant effects, which are attributed to different components. KEYWORDS: flavonoids, alkaloids, polysaccharides, essential oils, Citrus aurantium L. var. amara Engl., antioxidant

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fication of CAVA was performed by the corresponding author. The plant material was washed and dried at 60 °C in a hot air oven for 24 h. Then, the pretreated samples were ground to a fine powder and stored in sealed containers until needed. Chemicals and Reagents. DPPH, ABTS, 2,4,6-tri(2′-pyridyl)1,3,5-triazine (TPTZ), hesperidin, naringin, neohesperidin, synephrine, and acetonitrile were purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.). Preparation of Flavonoids, Alkaloids, Polysaccharides, Coumarins, and Essential Oils. Extraction procedures for total flavonoids, alkaloids, polysaccharides, coumarins, and essential oils from blossoms of CAVA were displayed in Figure 1. Initially, 200 g of ground powder was refluxed with 3000 mL of 80% ethanol solution for 3 h. After centrifugation and concentration, the resulting sample was loaded onto AB-8 macroporous resin and eluted with distilled water and 30% ethanol solution, successively. Eventually, 30% ethanol fractions were concentrated and collected as total flavonoids (CAVAF). As for total alkaloids, the extraction method was referred to Hu et al.,8 as follows: Dried powders of CAVA were extracted with ethanol solutions containing 2% HCl. Then, the sample was redissolved in 2% HCl solution, and its pH was adjusted to 2−3. After centrifugation, the supernates were extracted with chloroform twice and the fractions in chloroform phase were discarded to remove unnecessary substances. Thereafter, ammonia−water was added to the residual solution to adjust the pH at 9−10, followed by extraction with chloroform 4 times. The total alkaloids (CAVAA) were eventually obtained after the extracts in the chloroform phase were combined and evaporated. The crude polysaccharides (CAVAP) were obtained after refluxed with hot water, decolorized with D354FD resin, deproteinized with sevage reagent, and precipitated with anhydrous ethanol, successively. The detailed procedures for CAVAP preparation were demonstrated in our published report.7 Furthermore, ultrasound-assisted extraction was employed to extract the total coumarins. Blossoms of CAVA were initially extracted with 95% ethanol solution for 45 min with a ultrasonic cleaner and then further purified using the HPD300 macroporous resin eluted with

INTRODUCTION Reactive oxygen species at low levels were essential for life; however, excessive amounts could cause severe damage to health. Therefore, research on naturally derived antioxidants, especially those with greater efficacy and lower cytotoxicity, has attracted considerable interests. Many detection methods have been built to evaluate antioxidant effects. For example, an online high-performance liquid chromatography (HPLC) free radical scavenging detection system was a new technique.1 Specifically, 1,1-diphenyl-2-picrylhydrazyl radical (DPPH•), 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt radical cation (ABTS• +), hydroxyl radical (•OH), ferric-reducing antioxidant power (FRAP), and reducing power assays were frequently employed. Accumulative evidence suggested that flavonoids from plants were excellent antioxidants.2 Also, some polysaccharides were reported to exert pronounced antioxidant capability by electron or hydrogen donation or carbohydrate−hydroxyl radical complex formation.3 Alkaloids also had various physiological and pharmacological effects, including antioxidant effects.4 There were also published data confirming the antioxidant activities of Citrus coumarins and essential oils.5,6 Citrus aurantium L. var. amara Engl. (CAVA), belonging to Citrus, was traditionally used as an edible and medicinal material in China because of its various pharmacological activities.7 The objective of the present study was to give a comprehensive prediction for the antioxidant efficacy of major ingredients from CAVA. Although there were many studies on the antioxidant activity of these components in different varieties, few studies were found to compare their antioxidant activities in the same species using different models. It is hoped that the investigation method might provide some insights for the evaluation of other plants.

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MATERIALS AND METHODS

Received: Revised: Accepted: Published:

Plant Material. Dried blossoms of CAVA, collected in Zhejiang, China, during April and May, were purchased from a traditional Chinese medicine market in Guangzhou, China. Botanical identi© 2017 American Chemical Society

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May 13, 2017 June 26, 2017 July 7, 2017 July 7, 2017 DOI: 10.1021/acs.jafc.7b02244 J. Agric. Food Chem. 2017, 65, 6087−6092

Letter

Journal of Agricultural and Food Chemistry

Figure 1. Extraction and separation procedures of total flavonoids, alkaloids, polysaccharides, coumarins, and essential oils from blossoms of CAVA. 20% ethanol solutions (containing 0.2% NaOH solution). The eluents by 20% ethanol solution were concentrated and obtained as the total coumarins (CAVAC). Additionally, extraction of essential oils from CAVA were performed on the basis of previous literature.9 Briefly, 100 g of fine powder of CAVA was mixed with 2000 mL of distilled water and extracted by steam distillation for 6 h. Then, the separate layer on the water was eluted 4 times by an equal volume of diethyl ether and dehydrated by anhydrous sodium sulfate to remove traces of moisture. Eventually, the neroli was collected and named as CAVAO. Determination of the Chemical Constituent Content. HPLC and the combination of HPLC and mass spectrometry (MS) methods were frequently used as powerful approaches for the identification of phytochemical components in plant extracts. HPLC analysis was conducted using a DIONEX Ultimate 3000 HPLC system (ThermoFisher, Waltham, MA, U.S.A.) equipped with a C18 column (Waters, Milford, MA, U.S.A.) and a variable ultraviolet (UV) wavelength detector. Initially, validation of the HPLC method was conducted on the basis of the limit of detection (LOD) and limit of quantification (LOQ), linearity range, accuracy, precision, and ruggedness, according to Zhang et al.1 Elution was carried out at room temperature under gradient conditions with a mobile phase consisting of acetonitrile (A) and water (B) (containing 0.1% sodium dodecyl sulfate and 0.1% phosphoric acid). The chromatographic conditions were set as follows: 0−15 min, 20% A; 15−25 min, 20−25% A; 25−30 min, 25−33% A; and 30−60 min, 33−35% A, at a flow rate of 1.0 mL/min. The polysaccharide content of CAVAP was detected using the phenol− sulfuric acid method.10 UV spectrophotometry (275.4 nm) was employed to determine the coumarin content of CAVAC.11 DPPH Radical Scavenging Activity Assay. DPPH• scavenging capacity was measured using a 96-well microtiter spectrophotometric method.12 Briefly, 20 μL of sample solution at 12.5−800 μg/mL was mixed with 180 μL of 150 μmol/L DPPH solution. Thereafter, the reaction mixtures were shaken vigorously for 30 s and incubated for 30 min to reach a steady state. The absorbance at 517 nm was measured. Vitamin C (vit C), an ideal antioxidant preventing oxidative damage to lipids and other macromolecules, was used as a positive control. The scavenging activity of the DPPH radical was calculated as follows: scavenging rate (%) =

ODcontrol − ODsample ODcontrol

× 100%

where ODcontrol was the absorbance of the control (distilled water instead of sample) and ODsample was the absorbance in the presence of different samples. ABTS Radical Scavenging Activity Assay. Trolox equivalent antioxidant capacity was evaluated spectrophotometrically by the ABTS• + cation decolorization assay based on the published report.13 The ABTS radical cation was prepared by mixing 7 mmol/L aqueous ABTS solution with an equal volume of 4.9 mmol/L potassium persulfate solution. Then, 20 μL of different sample concentrations were mixed with 180 μL of diluted ABTS solution. The absorbance was eventually taken at 734 nm, and the scavenging percentage of ABTS• + was calculated on the basis of the following equation: scavenging rate (%) =

ODcontrol − ODsample ODcontrol

× 100%

(2)

where ODcontrol was the absorbance of the control (distilled water instead of sample) and ODsample was the absorbance in the presence of different samples. Hydroxyl Radical Scavenging Activity Assay. •OH scavenging activity was determined by the Smirnoff method, with some modifications.14 A total of 50 μL of different tested samples was incubated with 50 μL of H2O2 (60 mmol/L), 50 μL of FeSO4 (9 mmol/L), and 50 μL of salicylic acid−ethanol (9 mmol/L) for 30 min at 37 °C. Then, the absorbance at 510 nm was detected, and •OH scavenging activity was expressed as follows: scavenging rate (%) =

ODcontrol − ODsample ODcontrol

× 100%

(3)

where ODcontrol was the absorbance of the control (distilled water instead of sample) and ODsample was the absorbance in the presence of different samples. FRAP Assay. The FRAP of all tested samples was estimated according to Benzie and Strain.15 Initially, the FRAP reagent was prepared by mixing 2.5 mL of 40 mmol/L HCl, 2.5 mL of 20 mmol/L FeCl3·6H2O, and 25 mL of 0.3 mol/L acetate buffer (pH 3.6). Thereafter, 20 μL of sample solution at 12.5−800 μg/mL was mixed with 180 μL of FRAP reagent. After incubation at 37 °C for 30 min in a water bath, the absorbance of the reaction mixture was detected at 593 nm. The antioxidant activity showed direct proportion to the absorbance, and the average absorbance was expressed as 1 mmol/L concentration of Fe(II) solution.

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DOI: 10.1021/acs.jafc.7b02244 J. Agric. Food Chem. 2017, 65, 6087−6092

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Journal of Agricultural and Food Chemistry

Figure 2. HPLC chromatograms of the active constituents in blossoms of CAVA. HPLC profiles of (A) CAVAF, (B) CAVAA, (C) hesperidin, (D) synephrine, (E) naringin, and (F) neohesperidin. Reducing Power Assay. The production of Perl’s Prussian bluecolored complex was used as an indicator of the reducing power.16 Briefly, 40 μL of different samples at various concentrations of 12.5− 800 μg/mL was mixed with 100 μL of phosphate buffer (0.2 M, pH 6.6) and 100 μL of potassium ferricyanide solution (1%, w/v). The mixtures were then reacted at 50 °C for 20 min. Then, 100 μL of trichloroacetic acid (10%, w/v) solution was added to stop the reaction immediately. After centrifugation, 100 μL of supernate was mixed with 100 μL of distilled water and 20 μL of ferric chloride solution (0.1%, w/v). At 10 min later, the 96 plates were shaken sufficiently and measured spectrophotometrically at 700 nm. Statistical Analysis. All data were the average values of at least three independent experiments and expressed as the mean ± standard deviation (SD). The statistical significant difference was determined using a one-way analysis of variance (ANOVA). p < 0.05 was considered statistically significant, and p < 0.01 indicated highly statistically significant.

in CAVAF were identified as hesperidin, naringin, and neohesperidin, with the relative percent contents of 7.03, 43.40, and 33.51%, respectively (panels A, C, E, and F of Figure 2), and synephrine represented more than half (50.56%) of CAVAA (panels B and D of Figure 2). The chemical structures of naringin, neohesperidin, hesperidin, and synephrine were shown in Figure 3. In fact, the percent contents of identified compounds in this study were a little different from the previous reports.17 One possible explanation would be that the levels of phytochemicals were related to various factors, such as environmental and agronomic conditions, harvest and food processing operations, and storage factors.18 Additionally, the polysaccharide content of CAVAP and the coumarin content of CAVAC were 43.5 and 24.7%, respectively. Free Radical Scavenging Activity on DPPH•. After incubation with DPPH for 30 min, CAVAF, CAVAA, and CAVAC, especially at maximum concentrations, all showed visually noticeable discoloration from purple to yellow. CAVAF displayed the most significant scavenging activity on DPPH• in a dose-dependent manner at 100−800 μg/mL, followed with CAVAA, CAVAC, CAVAP, and CAVAO. Specifically, CAVAF at 800 μg/mL showed 51.59% DPPH• scavenging activity, comparable to vit C at 200 μg/mL.

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RESULTS AND DISCUSSION Contents of Total Flavonoids, Alkaloids, Polysaccharides, and Coumarins. The HPLC method had low LOD (0.001−0.005 mg/mL) and LOQ (0.005−0.010 mg/mL) and excellent linearity range (10.00−200.00 mg/mL), recovery rate (93.41−111.23%), precision [relative standard deviation (RSD) < 2%], and reproducibility (RSD < 2%). The main compounds 6089

DOI: 10.1021/acs.jafc.7b02244 J. Agric. Food Chem. 2017, 65, 6087−6092

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Journal of Agricultural and Food Chemistry

Figure 3. Chemical structures of naringin, neohesperidin, hesperidin, and synephrine.

at 200 μg/mL, higher than that of A-PTR (74.97%) at 250 μg/ mL as reported by our laboratory.23 A-PTR was alkali extraction of polysaccharide (A-PTR) from Pleurotus tuberregium (Fr.) Sing., and its significant •OH scavenging effect was ascribed to the presence of arabinose. Consistently, our recent study found that two homogeneous polysaccharides purified from CAVAP both contained arabinose, suggesting that arabinose might play a critical role in CAVAP scavenging •OH. FRAP Assay. Figure 4D demonstrated that all samples showed some degree of reducing power to reduce the TPTZ− Fe(III) complex to the TPTZ−Fe(II) complex. However, the reducing power was much lower than that of vit C. The reducing power of tested samples followed the following order: CAVAF > CAVAC > CAVAA > CAVAP > CAVAO. At the concentration of 800 μg/mL, the FRAP values of CAVAF, CAVAA, and CAVAC were 704.64, 260.45, and 340.97 μM, respectively, much lower than that of vit C (2579.65 μM) at the same concentration point. Reducing Power Assay. CAVAF displayed a concentration-dependent profile of reducing power at 200−800 μg/ mL, and its maximum reducing power reached 0.4082, close to that of vit C at 200 μg/mL (Figure 4E). In comparison to the aforementioned results, the reducing power of all tested samples was much lower than their eliminating activities toward free radicals. One possible explanation was that the reducing power was mainly achieved by terminating the radical chain by donation of a hydrogen atom and, therefore, could not represent the metal ion chelation of tested samples. In support, Chu et al. reported that potatoes showed low reducing power while high scavenging activities toward active oxygen.24 In conclusion, the antioxidant activities of different chemical components varied significantly toward different sources of free radicals and oxidants. CAVAF showed the strongest antioxidant capacity among all of the tested samples, as observed in DPPH,

Previous reports19,20 showed that hesperidin, naringin, and neohesperidin exhibited significant antioxidant effects, indicating that hesperidin, naringin, and neohesperidin, which represented 83.94% of CAVAF, might be responsible for the potent DPPH• scavenging property of CAVAF. Free Radical Scavenging Activity on ABTS• +. The reducing activity of the samples followed the order as follows: CAVAA > CAVAF > CAVAC > CAVAO > CAVAP. CAVAF, CAVAC, and CAVAA showed significant ABTS• + scavenging activities. However, the ABTS• + scavenging effects of CAVAP and CAVAO were not statistically significant (p > 0.05). Specifically, the ABTS• + scavenging activity of CAVAA at 800 μg/mL exceeded that of vit C. On the basis of previous reports,21 synephrine might be the specific alkaloid responsible for the ABTS• + scavenging activity of CAVAA because it composed 50.56% of CAVAA. Free Radical Scavenging Activity on •OH. The hydroxyl radical, the most active among the reactive oxygen species, could react with all adjacent biomolecules, such as proteins, lipids, and carbohydrates, thus resulting in various diseases. As shown in Figure 4C, CAVAC only displayed slight scavenging activity on • OH. Unexpectedly, CAVAF and CAVAO promoted the generation of •OH. CAVAA at the concentrations of 25−100 μg/mL provided a significant scavenging effect against •OH in a dose-dependent manner. However, with a continuously increasing concentration, the scavenging activity decreased from 69.68 to 32.31%. Specifically, the maximum scavenging effect of CAVAA on •OH was determined to be 69.68%, while that of vit C was only 56.85%. The results showed that CAVAP had better ability to quench • OH than DPPH• and ABTS• +, indicating that the plentiful hydroxyls in CAVAP might convert free radicals into stable products by donating an electron or combining with the radical ions.22 The scavenging rate of CAVAP on •OH reached 76.01% 6090

DOI: 10.1021/acs.jafc.7b02244 J. Agric. Food Chem. 2017, 65, 6087−6092

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Journal of Agricultural and Food Chemistry

Figure 4. Scavenging effects of CAVAF, CAVAA, CAVAP, CAVAC, and CAVAO on (A) DPPH•, (B) ABTS• +, and (C) •OH and their (D) FRAP value and (E) reducing power. All experiments were run in triplicate, and data showed mean ± SD values. (∗) p < 0.05 and (∗∗) p < 0.01 compared to the control group.

Funding

FRAP, and reducing power assays. CAVAA provided stronger protection against ABTS• + and •OH than vit C at some concentration points. CAVAP displayed significant scavenging effects on •OH. Using multiple in vitro assays to investigate the antioxidant effects of different bioactive components helps to better understand the role of different chemical components and their contributions to antioxidant activities of the species, which will be beneficial to the evaluations of other plants.

This project was supported by the Science and Technology Project of Guangzhou City (201604020150). Notes

The authors declare no competing financial interest.

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Chun-Yan Shen Tian-Xing Wang Xi-Mei Zhang Jian-Guo Jiang*

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REFERENCES

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College of Food and Bioengineering, South China University of Technology, Guangzhou, Guangdong 510640, People’s Republic of China

AUTHOR INFORMATION

Corresponding Author

*Telephone: +86-20-87113849. Fax: +86-20-87113843. E-mail: [email protected]. ORCID

Jian-Guo Jiang: 0000-0002-3361-6149 6091

DOI: 10.1021/acs.jafc.7b02244 J. Agric. Food Chem. 2017, 65, 6087−6092

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Journal of Agricultural and Food Chemistry

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DOI: 10.1021/acs.jafc.7b02244 J. Agric. Food Chem. 2017, 65, 6087−6092