In Vitro Antioxidant Activity of the Anthocyanins in Sageretia theezans Brongn Fruit

Abstract

Antioxidant activities of the extract containing anthocyanins from fruit of Sageretia theezans Brongn were investigated in this work. The fruit of Sageretia theezans Brongn was treated with 0.001 mol·L^–1 HCl in a solid (g): liquid (mL) ratio 1:10 to give extract. Total anthocyanins content was 253.10 ± 2.31 mg in 100 g fruit. The anthocyanins in extract were determined as cyanidin-3-sophorose-5-glucoside (Cy-3-Sp-5-Glu), petunidin-3-(6′-malonyl)-glucoside (Pt-3-(6’-Mal)-Glu), malvinidin-3-glucosid (Mv-3-Glu), and peonidin-3-(6′-malonyl)-glucoside (Pn-3-(6’-Mal)Glu) in 0.38, 22.57, 44.32, and 30.86%, respectively, by high performance liquid chromatography-mass spectrometry method. Antioxidant activities of the anthocyanins in extract were evaluated by methods of ferric reducing antioxidant power, hydroxyl radical scavenging assay, and superoxide radical scavenging assay. The anthocyanins in extract exhibited obvious antioxidative activities. These results suggested that the fruit of Sageretia theezans Brongn could be considered as a good source of natural antioxidants.

Keywords:

• Antioxidant activity
• Anthocyanins
• Sageretia theezans Brongn
• Free radical

INTRODUCTION

Sageretia theezans Brongn is an evergreen tender shrub of the Rhamnaceae family wild spread in Asia and tropical areas of North America.^[1] In South China, Sageretia theezans Brongn grows in the forest or bush on the hills or mountain at elevation below 2100 meters. Its fruit is edible for the natives. It blooms during late autumn and early winter and its fruit is ripe from April to May. Extract from Sageretia theezans Brongn exhibited anti-bacterial activity and anti-fungal activity.^[2] The edible fruit of Sageretia theezans Brongn rich in anthocyanins is a potential source of natural pigments, but less attention has been paid to it.

Anthocyanins are water-soluble flavonoids commonly found in plant tissues, including leaves, stems, roots, flowers, and fruits, responsible for the red, blue, and purple color of most flowers and fruits.^[3,4] These properties make anthocyanins as attractive natural colorants.^[5] Anthocyanins are glycosylated derivatives of 3,5,7,3´-tetrahydroxyflavylium cation, i.e., anthocyanidin; this aglycon part of the flavonoid structure, is renamed to anthocyanin when glycosylated. Anthocyanins are glycosides of so far about 30 kinds of anthocyanidins which differ by the degree of hydroxylation and methoxylation, and more than 500 representatives have been described up until now.^[6] Among the anthocyanidins discovered from plants, delphinidin, cyanidin, malvidin, peonidin, petunidin, and pelargonidin occured frequently. The anthocyanidins exist in cell vacuole in equilibrium of four molecular species: the colored basic flavylium cation and three secondary structures, the quinoidal base(s), the carbinol pseudobase (syn chromenol or hemiacetal) and the chalcone pseudobase. When pH < 7, the anthocyanidins are very stable, but this stability reduced when the pH approaches 7, and is completely destroyed while pH > 7.^[7]

Anthocyanins had pro-health functions, such as antineoplastic, radiation protective, vasoprotective, vasotonic, anti-inflammatory, chemo- and hepato-protective.^[8–10] Anthocyanins possessed potent antioxidant properties,^[10,11] which is due to the electron deficiency of anthocyanin molecules conferring them with the ability to quench free radicals.^[12] Huge numbers of literatures were available in safety record, but anthocyanins had not been used in western medicine.^[13] With some drawbacks, such as low extraction percentages and their relative instability affected by physical and chemical factors during processing and storage, the applications for anthocyanins in the food, pharmaceutical, and cosmetic industries have been limited.^[14]

Many methods have been used to study the antioxidant activities of different compounds and foods in vitro. These include ferric reducing antioxidant power (FRAP),^[15–17] oxygen radical absorbance capacity (ORAC),^[18,19] electron spin resonance (ESR),^[20,21] 1-(2,6-dimethylphenoxy)-2-(3,4-dimethoxyphenethylamino) propane hydrochloride (DDPH) assay,^[22–24] hydroxyl radical scavenging activity,^[25,26] the 2,2´-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid; ABTS) method,^[27,28] hydrogen peroxide scavenging activity,^[29] and superoxide radical scavenging assay.^[26,30] The aim of this research was to extract and identify of anthocyanins in Sageretia theezans Brongn fruit, quantify the total anthocyanins content by using the pH-differential method, and evaluate the in vitro antioxidant activities by three different methods.

MATERIALS AND METHODS

Chemicals

2,4,6-Tri(2-pyridyl)-1,3,5-triazine (TPTZ), and 1,2,3-benzenetriol were purchased from Aladdin Industrial Corporation. The following chemicals and compounds were obtained from the Lantian Laboratory Equipment Limited Company (Nanning): sodium acetate trihydrate (CH₃COONa·3H₂O), glacial acetic acid (CH₃COOH), ferric chloride hexahydrate (FeCl₃·6H₂O), 1,10-phenanthroline monohydrate, hydrogen peroxide (H₂O₂; 30%), ferrous sulfate (FeSO₄·7H₂O), sodium phosphate monobasic dihydrate (NaH₂PO₄·2H₂O), sodium phosphate dibasic dodecahydrate (Na₂HPO₄·12H₂O), ascorbic acid (Vc), tris(hydroxymethyl)aminomethane (Tris), ammonia solution (H₅NO), potassium chloride, methanol, phosphoric acid, sodium chloride, sodium hydroxide, and ethanol absolute.

Plant Materials and Treatments

The Chinese Sageretia theezans Brongn fruits grown in Guangxi Zhuang Autonomous Region (China) were used in this study. The samples were collected in Long An County (Nanning) in April of 2012 and identified by Dr. Ni S.F. (School of Life Science, Northwest University, Xi’an, China). The fresh fruits were placed in a draughty laboratory for one day at 15–20ºC, until there were no tiny drops of moisture on the surface of the fruits. Fruits were stored at –20°C to reduce degradations until needed for analysis.

The whole Sageretia theezans Brongn fruits (500 g) were milled using a mortar. Milled fruits were added to 5.0 L 0.001 mol·L^–1 HCl, placed in a 40°C sonicator bath for 1 h, filtrated under reduced pressure condition. The remaining residue (including pericarp and seed) was extracted two additional times with the same concentration HCl (the volume were 3.0 and 2.0 L, respectively). For the latter two extractions, the mixture was treated under the same conditions. The filtrates were collected and pooled. The pooled filtrates were evaporated under reduced pressure at 40°C to approximately 1 L. Finally, the volume was adjusted to 1.0 L and the value of pH was 2. The solution obtained was used for quantification of total anthocyanins, purification by cation exchange resin, and high performance liquid chromatography-mass spectrometry (HPLC-MS) analysis. Besides, the pooled filtrates were placed in a vacuum freezing dryer (TF-FD-1) at –60°C for 24 h. The deep red viscous extractive was used to test antioxidant activities. Both the solution obtained from first method and the deep red viscous extractive were stored at 4°C to reduce degradations until needed for analysis.

Total Anthocyanins Content of the Samples

The total anthocyanins were determined using the pH-differential method.^[31] The absorbance was measured at 520 and 700 nm in buffers at pH 1.0 (0.025 mol·L^–1 potassium chloride buffer) and 4.5 (0.4 mol·L^–1 sodium acetate buffer). The absorbance of the samples was calculated using the relation A = (A_{520 nm} − A_{700 nm}) _{pH 1.0} − (A_{520 nm} − A_{700 nm}) _{pH 4.5}. The results were expressed as milligrams of cyanidin-3-glucoside equivalents per 100 g (cy-3-glc Eq mg/100 g).

where, A is the absorbance, ε is the cyanidin-3-glucoside molar absorbance (26,900 L mol^–1 cm^–1), L is the cell path length (1 cm), MW is the molecular weight of cyanidin-3-glucoside (449.2 g mol^–1), DF is the dilution factor, V is the final volume (mL), and W is the fresh weight (g).

Analysis of Anthocyanins and HPLC-MS Confirmation of Anthocyanins Identity

Separation and purification treatments of the solution obtained from first method have been carried out using the column packed with a cation exchange resin (732) in hydrogen ion form. Resin activation was performed with 4–6 wt% HCl for 4 h then 2–5% wt% sodium hydroxide also for 4 h, finally 4–6 wt% HCl. After these procedures, deionized water was used to wash and elute resin beds. The separation of anthocyanin components has been carried out as follows: First, 500 mL of the solution (pH = 2) obtained from the first method were passed through the cationic exchange column with pump flow rate of 3 Vb·h^–1, column temperature 35°C and 1 h for completely adsorbed. Then, the column was thoroughly washed with deionized water, and eluted with 70% ethyl alcohol-water (v/v, containing 0.003 mol·L^–1 HCl) with the gradient flow rate of 10, 8, 6, 4, and 2 Vb·h^–1 to recover the anthocyanins. After concentration, the volume of eluates was adjusted to 500 mL and the value of pH was 2.

The eluates were separated and purified with Waters 2695 HPLC system equipped with an XBridge^TM C18 column (5 μm, 4.6 × 250 mm, Waters), and a Waters 2998 PDA UV-vis detector. The column was eluted with a mobile phase (containing 0.5 mol·L^–1 phosphoric acid) consisting of methanol:water (60:40, v/v) with flow rate of 0.8 mL·min^–1. Samples (pH 2) were subjected to passing through a 0.22 μm filter prior to HPLC analysis. The injection volume was 25 μL. The separated anthocyanins were detected and measured at 520 nm, and the identity of anthocyanins was based on the congruence of retention time. Operating parameters of the mass spectrometer were capillary temperature 350°C; spray needle voltage set at 4.50 kV. Nitrogen was used as a sheath gas.

FRAP Assay

In our study, total antioxidant activity was assayed with the original method of Benzie and Strain^[15] where some modifications were also used. The FRAP assay was based on the ability of the antioxidant to reduce ferric ion (Fe³⁺) to ferrous ion (Fe²⁺) in the presence of TPTZ, forming an intense blue Fe²⁺-TPTZ complex with an absorbance maximum at 593 nm, which is pH-dependent. FRAP reagent was prepared from 300 mmol·L^–1 acetate buffer (pH 3.6), 10 mmol·L^–1 TPTZ in 40 mmol·L^–1 HCl and 20 mmol·L^–1 ferric chloride in proportions of 10:1:1 (v/v) just before use and heated to 37°C. The 300 mmol·L^–1 acetate buffer was prepared by mixing 3.1 g of sodium acetate trihydrate (CH₃COONa·3H₂O) with 16 mL glacial acetic acid and brought to 1 L with deionized water. Different concentrations (50, 120, 190, 260, and 330 μg·mL^–1) of sample (the deep red viscous extractive) solution (0.5 mL) were mixed with FRAP reagent (3.0 mL), and 5.0 mL deionized water was added. The reaction mixture was incubated at 37°C for 30 min and the absorbance was measured at 593 nm by UV-vis spectrophotometer (722). Ascorbic acid was subjected to the same treatment except for the concentrations (5, 10, 15, 20, and 25 μg·mL^–1). The results were calculated from the standard curves prepared using different concentrations (0.04–1.00 mmol·L^–1) of ferrous sulfate expressed as mmol Fe²⁺/L.

Hydroxyl Radical Scavenging Activity Assay

The hydroxyl radical scavenging activity was determined using the method described by Jin, Cai, Li, and Zhao^[32] with slight modifications. The sample group was 1 mL various concentration sample (100, 200, 500, 1000, 1500, 2000, and 2500 μg·mL^–1), 2 mL 1,10-phenanthroline (0.75 mmol·L^–1), 2 mL phosphate buffer (0.75 mmol·L^–1, pH 7.4), and 2 mL ferrous sulfate (0.75 mmol·L^–1) being mixed thoroughly. Then 1 mL hydrogen peroxide (0.01%) was added. The mixture was incubated at 37°C for 60 min. Afterward, the absorbances of the mixture were measured at 536 nm. The damaged group contained the same solutions as the sample group, except for using 1 mL deionized water instead of the sample. The non-damaged group contained the same solutions as the damaged group except for using 1 mL deionized water instead of hydrogen peroxide. Results were determined according to the following equation:

where, As, Ad, and An represented the absorbance of the sample, the damaged and non-damaged groups, respectively. The percentage of the radical scavenging activity of the extract was plotted against the sample concentration to calculate the IC₅₀, which is the amount of extractive necessary to reach the 50% radical scavenging activity. Commercial ascorbic acid (100, 200, 300, 400, and 500 μg·mL^–1) was used as reference standards.

Superoxide Anion Radical Scavenging Activity Assay

Superoxide anion radical scavenging activity was carried out as per the method of Zhang et al.^[33] with slight modifications. Deionized water (0.2 mL), and 5.4 mL Tris-HCl (pH 8.0, 0.05 mol·L^–1) were mixed thoroughly. The mixture was incubated at 25°C for 10 min. Then 0.4 mL preheated (25°C) 1,2,3-benzenetriol (30 mmol·L^–1) was added to the mixture quickly. Afterward, the absorbances of the mixture were measured at 420 nm every 30 s, lasting for 5 min. The absorbance was recorded as A₁. Two milliliters of different concentrations extract (50, 200, 400, 800, 1200, and 1600 μg·mL^–1) or ascorbic acid (100, 200, 300, 400, and 500 μg·mL^–1), and 5.4 mL Tris-HCl (pH 8.0, 0.05 mol·L^–1) were mixed thoroughly. The mixture was incubated at 25°C for 10 min. Then 0.4 mL HCl (10 mmol·L^–1) was quickly added to the mixture. The absorbance (A₂) of the mixture was measured the same to A₁. Sample group contained the same solutions as the test of A₂ except using 0.4 mL preheated (25°C) 1,2,3-benzenetriol (30 mmol·L^–1) instead of 0.4 mL HCl. The absorbance was recorded as A₃. The percentage of scavenging superoxide anion of the extract or ascorbic acid was calculated as follows:

Commercial ascorbic acid (100, 200, 300, 400, and 500 μg·mL^–1) was used as reference standards.

Statistical Analysis

All samples were prepared and analysis in triplicate. Values are expressed as means ± standard deviations of triplicate determinations. The correlation coefficient (R²) was used to show correlation. Analysis of variance was used to determine the significance (p ≤ 0.05) of the data obtained in all experiments. All results were determined to be within the 95% confidence level for reproducibility.

RESULTS AND DISCUSSION

HPLC-MS Analysis

Total anthocyanins content from the Sageretia theezans Brongn fruits as quantified by the pH-differential method was 253.10 ± 2.31 mg/100 g. As shown in Fig. 1, five peaks were observed from the HPLC results, which represented five types of anthocyanins in Sageretia theezans Brongn fruit. The retention time and relative content of these anthocyanins are shown in Table 1. The MS results (Fig. 2) further confirmed the detected peaks.

TABLE 1 Relative contents of five anthocyanins and their retention time

Peak number	Retention time (min)	Relative content %
Peak 1	2.77	0.38
Peak 2	2.96	22.57
Peak 3	3.41	1.69
Peak 4	4.28	44.32
Peak 5	6.16	30.86

FIGURE 1 HPLC profile of Sageretia theezans Brongn fruit.

FIGURE 2 MS profile of (A) compound 1; (B) compound 2; (C) compound 3; and (D) compound 4.

FIGURE 2 Continued.

Compound 1 (retention time 2.77) showed a molecular ion peak at m/z = 773.98, and other fragment ion peaks at m/z = 277.10, and 611.92. Daughter ion peaks were at m/z = 287 and m/z = 331, and they were the fragment ions of cyanidin and sophorose, respectively. According to Liu et al.,^[34] compound 1 was confirmed as cyanidin-3-sophorose-5-glucoside. And the content was 0.96 ± 0.01 mg/100 g.

Molecular ion peak of compound 2 (retention time 2.96 min) appeared at m/z = 564.93. Other fragment ion peak appeared at m/z = 478.91. Product ion scan showed that m/z = 317 and 248 were the fragment ions of petunidin and malonyl glucosyl. Compound II was identified as petunidin-3-(6′-malonyl)-glucoside, according to Gao et al.^[35] Its content was 57.12 ± 0.52 mg/100 g.

Based on the analysis of the [M + H]⁺ ion peak ([m + 1]/z = 494.86), the fragment ion peaks of malvinidin (m/z = 331) and glucosyl (m/z = 162), and combining with Ding et al.,^[36] compound 3 was determined to be malvinidin-3-glucoside. Its content was 112.12 ± 1.02 mg/100 g.

The data of the molecular ion peak (m/z = 549) and the fragment ion peaks (m/z = 463, 162) suggested that peonidin and malonyl glucosyl were connected to compound 4. Referring to Ding et al.^[36] and Liu et al.,^[37] compound 4 was recognized as peonidin-3-(6′-malonyl)-glucoside. The content was 78.11 ± 0.71 mg/100 g.

FRAP Assay

The FRAP assay appears to be an attractive and potentially useful assay among all the methods used for the measurement antioxidant defense system. The FRAP assay is quick and easy to conduct. The reaction is linearly related to a wide concentration range of the antioxidants present.^[15]

The calibration curve (Fig. 3A) revealed a highly positive linear (R² = 0.9990) relation between A (593 nm) and concentration of ferrous sulfate standards. This curve was, therefore, employed to reliably estimate antioxidant potential of the anthocyanins sample and ascorbic acid. Five concentrations were tested in this assay for the sample and ascorbic acid, and they were 50, 120, 190, 260, 330 μg·mL^–1 and 5, 10, 15, 20, 25 μg·mL^–1, respectively. Figure 3B depicted the FRAP values of anthocyanins sample and ascorbic acid, and they had a highly positive linear (R² = 0.9997 and 0.9991, respectively) relation with their concentrations. Results indicated that ascorbic acid had higher antioxidant activity in relatively lower concentrations when compared to anthocyanins sample. The probable reason was that the impurity in anthocyanins sample affecting activity. But the anthocyanins in Sageretia theezans Brongn fruit exhibited obvious antioxidant activity in the FRAP assay. The FRAP value 0.634 (mmol·L^–1) could be expressed as 192.12 mmol FeSO₄/100 g, and was greater than that of strawberry anthocyanins.^[38]

FIGURE 3 (A) Calibration curve of ferrous sulfate standards (0.04, 0.08, 0.12, 0.16, 0.20, 0.24, 0.28, 0.32, 0.36, and 1.00 mmol·L^–1), n = 3. Y = 1.2838X + 0.0229 and R² = 0.9990; (B) FRAP values of different concentrations of the anthocyanins sample and ascorbic acid, n = 3.

Hydroxyl Radical Scavenging Activity Assay

Hydroxyl radicals were generated by Fenton reaction in the system of H₂O₂-Fe²⁺. The reaction of ferrous ions with 1,10-phenanthroline generated a stable orange complex which was the basis for the classical spectrophotometric method. The orange complex had an absorption maximum at 536 nm. When the ferrous ions was oxidized to ferric ions by hydroxyl radicals, the absorption of the complex at 536 nm disappeared. While hydroxyl radical scavenger was added to the reaction system, this process was restrained and the absorption at 536 nm did not disappear completely. The activity of scavenging hydroxyl radical was calculated, illustrated in Fig. 4.

Both two curves (Fig. 4) revealed a highly positive linear relation between scavenging activity and concentration of the tested samples. Moreover, as shown in Fig. 4, IC₅₀ of anthocyanins and ascorbic acid were calculated as 1577.67 and 319.77 μg·mL^–1, respectively, and suggested that the hydroxyl radical scavenging activity of highly purified ascorbic acid was higher than that of non-purified anthocyanins sample. The activity of purple sweet potato anthocyanins (52.5% at 200 μg·mL^–1)^[39] was higher than anthocyanins samples (52.5% at 1984 μg·mL^–1). Anthocyanins was stable at pH < 7, and this stability was reduced when the pH ≥ 7.^[7] The impurity in anthocyanins extractive strongly affected the activity of scavenging hydroxyl radical.

FIGURE 4 The hydroxyl radical scavenging activity of anthocyanins and ascorbic acid, n = 3.

Superoxide Anion Radical Scavenging Activity Assay

1,2,3-Benzenetriol autoxidizes rapidly, particularly in alkaline solution. Self-oxidation of 1,2,3-benzenetriol is a chain reaction, with superoxide anion radical generating. The reaction product is colored and can be measured by spectrophotometry at 420 nm. Self-oxidation of 1,2,3-benzenetriol is inhibited when antioxidant was added to the reaction system. The degree of inhibition rate reflects the scavenging activity of superoxide anion radical.^[32] Superoxide anion radical scavenging activities of anthocyanins sample and ascorbic acid are shown in Tables 2 and 3. The scavenging activities increased with the increase of the concentrations. Scavenging activities reached maximum 58.47% at 1600 μg·mL^–1 for anthocyanins sample, 73.08% at 500 μg·mL^–1 for ascorbic acid. The antioxidant activity of anthocyanins in Sageretia theezans Brongn fruits was less than that of anthocyanins in purple sweet potato.^[39] Results indicated that scavenging activity decreased when time increased. The cause was probably that the self-oxidation of 1,2,3-benzenetriol generated an unstable semiquinone radical and a superoxide anion.^[40] The delocalization in the conjugated system produced by anthocyanins and superoxide anion stabilized the semiquinone radical, and enhanced the absorption at 420 nm.^[41] This delocalization effect was weak for ascorbic acid. So its decrease of scavenging activity was smaller than anthocyanins sample.

TABLE 2 Superoxide anion radical scavenging activity of anthocyanins at different time, n = 3

Concentration (μg·mL^−1)		Time (min)
		0.5	1.0	1.5	2.0	2.5	3.0	3.5	4.0	4.5	5.0
50	Scavenging activity (%)	46.61	33.33	23.45	20.45	18.58	16.51	15.59	15.56	14.29	14.06
200		48.31	33.90	26.55	23.86	20.95	19.63	18.24	17.50	16.17	15.89
400		49.15	33.90	26.55	22.73	20.27	18.07	17.35	16.67	15.36	15.10
800		51.69	37.85	29.65	23.86	21.62	19.00	17.94	17.50	16.17	15.36
1200		55.08	34.46	26.99	22.73	19.93	17.76	15.88	16.11	14.82	14.32
1600		58.47	38.98	30.09	25.38	22.30	20.25	18.82	18.61	16.71	16.93

TABLE 3 Superoxide anion radical scavenging activity of ascorbic acid at different time, n = 3

Concentration (μg·mL^–1)		Time (min)
		0.5	1.0	1.5	2.0	2.5	3.0	3.5	4.0	4.5	5.0
100	Scavenging activity (%)	25.64	24.81	18.86	15.24	14.11	13.16	11.31	11.30	9.94	9.88
200		35.90	32.33	25.71	23.33	21.58	19.55	18.02	17.61	16.35	16.05
300		52.56	46.62	40.57	35.71	32.78	30.83	28.27	26.91	25.32	24.38
400		64.10	55.64	49.14	44.76	42.32	39.85	36.04	34.55	32.37	30.86
500		73.08	66.17	61.14	55.71	52.28	49.25	45.94	44.19	41.35	39.81

CONCLUSION

In our study, the total anthocyanins content of Sageretia theezans Brongn fruit was quantitated as 253.10 ± 2.31 mg/100 g by using the pH-differential method. HPLC-MS separation and identification indicated, for the first time, the presence of cyanidin-3-sophorose-5-glucoside, petunidin-3-(6’-malonyl)-glucoside, malvinidin-3-glucoside, and peonidin-3-(6′-malonyl)-glucoside in the edible fruit of Sageretia theezans Brongn. The contents were 0.96 ± 0.01, 57.12 ± 0.52, 112.12 ± 1.02, and 78.11 ± 0.71 mg/100 g, respectively. But they were not the accurate contents for lack of the standards in HPLC analysis. The results showed the extract from Sageretia theezans Brongn fruit exhibited attractive in vitro chemical antioxidant activity. Anthocyanins of Sageretia theezans Brongn fruit could be used as an addition for food, cosmetics, and beverages or drinks.

FUNDING

This work was supported by the National Natural Science Foundation of China [81060261], and the Natural Science Foundation of Key Projects of Guangxi Province, China [2011GXNSFD018016].

Additional information

Funding

This work was supported by the National Natural Science Foundation of China [81060261], and the Natural Science Foundation of Key Projects of Guangxi Province, China [2011GXNSFD018016].