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Antioxidant capacity of anthocyanins from Rhodomyrtus tomentosa (Ait.) and identification of the major anthocyanins
Accepted Manuscript Antioxidant capacity of anthocyanins from Rhodomyrtus tomentosa (Ait.) and identification of the major anthocyanins Chun Cui, Shaomin Zhang, Lijun You, Jiaoyan Ren, Wei Luo, Wenfen Chen, Mouming Zhao PII: DOI: Reference:
S0308-8146(13)00142-8 http://dx.doi.org/10.1016/j.foodchem.2013.01.107 FOCH 13656
To appear in:
Food Chemistry
Received Date: Revised Date: Accepted Date:
30 October 2012 5 January 2013 28 January 2013
Please cite this article as: Cui, C., Zhang, S., You, L., Ren, J., Luo, W., Chen, W., Zhao, M., Antioxidant capacity of anthocyanins from Rhodomyrtus tomentosa (Ait.) and identification of the major anthocyanins, Food Chemistry (2013), doi: http://dx.doi.org/10.1016/j.foodchem.2013.01.107
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1
Antioxidant capacity of anthocyanins from Rhodomyrtus tomentosa (Ait.) and
2
identification of the major anthocyanins
3
Chun Cui1, Shaomin Zhang1, Lijun You1, Jiaoyan Ren1, Wei Luo2, Wenfen Chen1, Mouming
4
Zhao1,*
5
1
College of Light Industry and Food, South China University of Technology, Guangzhou
6 7
510640, China 2
Analysis and Testing Center, South China University of Technology, Guangzhou 510640,
8
China
9 10 11
Corresponding author
12
Mouming Zhao, Professor
13
Tel/Fax: +86 20 87113914
14
E-mail: [email protected]
1
15
ABSTRACT
16
The anthocyanins in the fruits of Rhodomyrtus tomentosa (ACN) were extracted by 1%
17
TFA in methanol, and then purified by X-5 resin column and C18 (SPE) cartridges. The
18
purified anthocyanin extract (ART) from the fruits of Rhodomyrtus tomentosa showed strong
19
antioxidant activities, including DPPH radical-scavenging capacity, ABTS radical scavenging
20
capacity, reducing power and oxygen radical absorbance capacity (ORAC). The purified
21
anthocyanin extract was analyzed by high performance liquid chromatography (HPLC). The
22
major anthocyanins were purified by semi-preparative HPLC and Sephadex LH-20 column
23
chromatography, and were identified as cyanidin-3-O-glucoside, peonidin-3-O-glucoside,
24
malvidin-3-O-glucoside,
25
pelargonidin-3-glucoside by HPLC-ESI/MS and nuclear magnetic resonance spectroscopy
26
(NMR). Cyanidin-3-O-glucoside was considered as the most abundant anthocyanin, which
27
was 29.4 mg/100 g dry weight of R. tomentosa fruits. Additionally, all the major anthocyanins
28
were identified from R. tomentosa fruit for the first time.
29 30
Keywords:
petunidin-3-O-glucoside,
Rhodomyrtus
tomentosa;
Cyanidin-3-O-glucoside
2
delphinidin-3-O-glucoside
Anthocyanins;
Antioxidant
and
activity;
31
1. Introduction
32
Rhoddmyrtus tomentosa (Ait.) Hassk, a member of the Myrtaceae family, commonly known
33
as rose myrtle, is an abundant evergreen shrub native to southeast Asia, with rose-pink flowers
34
and dark-purple edible bell-shaped fruits (Amporn, Tony, & John, 2005). The stem, leaf, fruits
35
of the whole plant can be used as medical materials. The R. tomentosa fruit possesses
36
excellent pharmacological properties, including antibacterial activity against Gram-positive
37
bacteria, such as Streptococcus pyogenes and Escherichia coli (Dachriyanus et al., 2002;
38
Surasak & Supayang, 2008).
39
Rhodomyrtus tomentosa fruit is widely distributed in south China; its bright purplish-red
40
colour is due to anthocyanins. Anthocyanins, an important group of water-soluble pigments in
41
natural products, are widely spread in flowers, fruits and leaves. They usually link with sugar
42
moieties and constitute flavonoids, attracting more and more attention due to their usage as
43
natural food additives and excellent functional properties for human health (Kaliora,
44
Dedoussis, & Schmidt, 2006; Li, Wang, Guo, & Wang, 2011). On the basis of their structural
45
characteristics, anthocyanins possess various biological activities, including antioxidant
46
(Cerezo, Cuevas, Winterhalter, Garcia-Parrilla, & Troncoso, 2010), anticancer (Wang & Stoner,
47
2008), anti-inflammatory (Greenspan, Bauer, Pollock, Gangemi, Mayer, & Ghaffar, 2005),
48
anti-artery atherosclerosis, anti-hypertensive (Pinent, Blay, Bladé, Salvadó, Arola, & Ardévol,
49
2004) and antibacterial activities (Lacombe, Wu, Tyler, & Edwards, 2010). In recent decades,
50
the antioxidant activities of anthocyanin and its working mechanism have attracted growing
51
global interest. As reported, anthocyanin might play its protective role through the working
52
system of H atom transfer, single electron transfer and metal chelation (Monica, Nino, &
53
Marirosa, 2011). However, to our knowledge, the information regarding the antioxidant
54
capacity and major anthocyanins of Rhodomyrtus tomentosa is limited.
55
The objectives of the present study were to extract and purify the anthocyanins from the 3
56
fruit of R. tomentosa and to evaluate their antioxidant capacity. The major anthocyanins were
57
further isolated by semi-preparative HPLC and column chromatography, and identified by
58
HPLC-ESI-MS and NMR spectroscopy.
59
2. Materials and methods
60
2.1. Plant material
61
The wild-grown mature fruits of Rhodomyrtus tomentosa (Ait.) Hassk were collected in
62
Shanwei, Guangdong Province, China, in August (Fig. 1), freeze-dried after being washed
63
with clean sterile water, and then stored at -20oC prior to extraction.
64 65
2.2. Chemicals
66
2,2´-Azobis
(2-methylpropionamidine)
dihydrochloride
(AAPH),
2,2-diphenyl-1-
67
picrylhydrazyl (DPPH), 2,2´-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid) (ABTS),
68
6-hydroxy-2,5,7,8-tetramethyl-2-chromanecarboxylic acid (trolox), sodium fluorescein, 3´,6´-
69
dihydroxyspiro [isobenzofuran-1[3H],9´[9H]-xanthen] -3-one(FL), ascorbic acid, CF3COOD
70
and CD3OD were purchased from Sigma Chemical Co. (St. Louis, MO,USA). X-5 resins were
71
obtained from Haiguang Chemical Co. Ltd., (Tianjin, China). Sephadex LH-20 was purchased
72
from Pharmacia Fine Chemicals Co. (Uppsala, Sweden) and Sep-Pak cartridges were
73
purchased from Waters Co., (Milford, Bedford, MA, USA). All the solvents for HPLC
74
analysis were of HPLC grade. All the other chemicals used were of analytical grade.
75 76
2.3. Extraction of anthocyanins
77
Free-dried sample (300 g) was macerated with 1000 ml of TFA (trifluoroacetic acid):
78
methanol (1:99; v/v) for 48 h in the dark at room temperature, and the remaining residues were
79
extracted by 400 ml of the extract solvent to extract anthocyanin. The supernatants were
80
obtained by centrifugation (8,000 × g, 15 min) in a GL-21M refrigerated centrifuge (Xiangyi 4
81
Instrument Co. Ltd., Changsha, China) and filtration. Finally, the acidic methanol extracts
82
were combined and evaporated, using a rotary evaporator (RE-52AA, Yarong Instrument
83
Factory, Shanghai, China) at 40 °C.
84 85 86
2.4. Purification of anthocyanins The concentrated crude extract was purified by partition (several times) against ethylacetate
87
and chloroform to remove non-polar compounds.
88
time) phase was then subjected to a X-5 resin column (1.6 × 40 cm) to remove free sugars,
89
aliphatic acids and other water-soluble compounds by washing (several times) with the
90
column volume of distilled water. The adsorbed anthocyanins were eluted using methanol
91
containing 0.1% trifluoroacetic acid (TFA, v/v).
The partial purified aqueous (10 ml each
92
The concentrated anthocyanin extract was further refined by solid phase extraction (SPE) in
93
C18 cartridges (Sep pak, Waters). The aqueous extract of anthocyanin was passed through a
94
sorbent C-18 Sep-Pak cartridge (Waters Associates, Milford, MA) previously activated with
95
acidified methanol (0.01% HCl v/v) and equilibrated with water. The extract adsorbed onto
96
the cartridge was rinsed with ultra-pure water to remove water-soluble impurities, and then
97
eluted with acidified methanol (0.01% HCl, v/v). The acidified methanol solution was
98
evaporated under vacuum, redissolved in water, and lyophilized (R2L-100KPS, Kyowa
99
Vacuum Engineering, Tokyo, Japan). The dry fraction was dissolved with deionized water.
100
Samples were filtered through a 0.45 μm filter before analysis.
101 102
2.5. Isolation and identification of the main anthocyanins from the purified extract
103
2.5.1. Isolation
104
The purified extract, containing the major anthocyanin-derived pigments, was isolated by
105
semi-preparative HPLC, using a Waters X-bridge reversed-phase C18 column (5μm, 10 × 150 5
106
mm, i.d.) at 35 °C with a flow rate of 4.5 ml/min and was monitored at 520 nm. The solvents
107
were (A), water/formic acid (98:2), and (B), formic acid/methanol (2:98), with the following
108
gradient: 10% to 15% B over 5 min, 15% to 18% B over 5min, 18% to 23% B over 20 min,
109
from 23% to 25% B over 10 min. and then each fraction was further chromatographed on a
110
Sephadex
111
methanol/water/trifluoroacetic acid at a ratio of 20:79.5:0.5 to obtain compounds 1 to 6 ,
112
respectively.
LH-20
column
(1.0
×
60
cm),
eluting
with
a
mixture
of
113 114
2.5.2. Molecular weight determination
115
A MS system (Esquire HCT PLUS, Phenomenex, Torrance, CA, USA), equipped with a
116
Hewlett–Packard1100 series liquid chromatography system, was used to determine the
117
molecular weight of each compound. One hundred microlitres of sample solution (100 μg/ml)
118
was injected into the MS system. Mass spectra in the positive-ion mode were generated under
119
the following conditions: HV capillary = 2800 V; HV end plate offset = -500V; nebulizer
120
pressure = 10 psi; dry temperature = 300°C; Dry Gas = 5.00 l/min; m/z range = 100–1000. A
121
reversed-phase column (250 × 4.6 mm, 5 μm, C18), thermostatted at 35 °C, was used for
122
separation; solvents were (A) aqueous 0.1% trifluoroacetic acid, and (B) 100% acetonitrile,
123
establishing the gradient as described in 2.10.
124 125 126
2.5.3. NMR identification The NMR results were obtained at 400 MHz and 100 MHz for 1H and 13C, respectively, on
127
a Bruker AVANCE Spectrometer (Bruker DRX400, Bruker Biospin Co., Karlsruhe, Germany)
128
in the solvent, CF3COOD-CD3OD (5:95; v/v); coupling constants were expressed in Hertz,
129
and chemical shifts were given on a δ (ppm) scale with TMS (tetramethylsilane) or solvent
130
signals as an internal standard. 6
131 132
2.6. Determination of total anthocyanin content (TAC)
133
TAC was determined according to the pH differential method by Kim, Jeong and Lee
134
(2003). Absorbance was measured at 520 and 700 nm in buffers at pH 1.0 and 4.5, using A =
135
[(A520 - A700)pH 1.0 - (A520 - A700)pH 4.5] and expressed as mg of cyanidin-3-glycoside (molar
136
extinction coefficient of 26900 and molecular weight of 449.2) equivalents per 100 g of dry
137
fruit weight. TAC was calculated using the following equation. Data were reported as means ±
138
standard deviation of triplicate determinations.
139
TAC (mg /100g) = A × MW × DF ×
1 V × ×100 ε×L M
(1)
140
where A is absorbance, ε is cyanidin-3-O-glucoside molar absorbance (26900), L is the cell
141
pathlength (1 cm), MW is the molecular weight of cyanidin-3-glucoside (449.2 Da), DF is the
142
dilution factor, V is the final volume (ml), and M is the dry weight (mg).
143 144
2.7. DPPH radical-scavenging activity assay
145
DPPH radical-scavenging activity was measured according to the method of Wu, Chen, and
146
Shiau (2003) with a slight modification. Aliquots (2.0 ml) of 0 (control), 2, 4, 6, 8, 10, 12, 20
147
and 30 μg/ml of anthocyanin extract (ART) dissolved in distilled water were added to 2.0 ml
148
of 0.2 mM DPPH• that was dissolved in 95% ethanol. The mixture was then shaken
149
vigorously using a mixer (QT-1 Mixer, Tianchen Technological Co.Ltd., Shanghai, China).
150
The reaction mixture was incubated for 30 min at 30°C in the dark. The absorbance of the
151
resulting solution was recorded at 517 nm by a spectrophotometer (UV2100, Unico Instrument
152
Co., Ltd., Shanghai, China). The scavenging activity was calculated using the following
153
equation:
154
Scavenging
activity
(%)
＝
(ADPPH•
sample
155
－ Asample
control)
×
100/ADPPH•
blank
(2 7
156
)
157
where ADPPH• sample = absorance of 2 ml of sample solution + DPPH solution; Asample control =
158
absorance of 2 ml of sample solution + 2 ml of 95% ethanol; and ADPPH• blank = absorance of 2
159
ml of 95% ethanol + DPPH• solution. Ascorbic acid was used as the reference. IC50 value (μg
160
compound ml-1), the concentration of extract that was required to scavenge 50% of radicals,
161
was calculated.
162 163
+ 2.8. ABTS • radical cation-scavenging activity assay
164
ABTS radical cation-scavenging activity of anthocyanins was determined as described by
165
+ Wang and Xiong (2005) with a slight modification. The ABTS• solution was prepared with
166
+ final concentrations of 7 mM ABTS• and 2.45 mM potassium persulfate. The solution was
167
incubated for 16 h at room temperature in the dark until the reaction was completed. Prior to
168
+ the assay, the absorbance of the ABTS• solution at 734 nm was adjusted to 0.70 ± 0.02 by
169
dilution with 0.2 M sodium phosphate-buffered saline (pH 7.4). Then 40 μl of ART (30, 50,
170
+ 80, 100, 120, 160, 200 μg/ml) were added to 4 ml of diluted ABTS• solution. The mixture
171
was shaken vigorously for 30 s and allowed to stand in the dark for 6 min. An equivalent
172
volume of distilled water, instead of the sample, was used for the blank. The absorbance of the
173
resultant solution was measured at 734 nm. A standard curve was obtained by using trolox
174
standard solution at various concentrations (0.4, 0.8, 1.2, 1.6, 2.0, 2.4 mM) with ethanol. The
175
standard curve was prepared by reacting 40 μl of trolox (0.4, 0.8, 1.2, 1.6, 2.0, 2.4 mM) with 4
176
+ ml of diluted ABTS• solution. The degree of ABTS radical-scavenging activity of
177
anthocyanins was calculated, based on the trolox standard curve, and was expressed in terms 8
178
of mg trolox equivalents (TE) /mg anthocyanin.
179 180
2.9. Reducing power assay
181
The reducing power of anthocyanins was determined according to the method of Oyaizu
182
(1988) with a slight modification. ART were dissolved in distilled water to obtain various
183
concentrations (10, 20, 40, 60, 80, 100 μg/ml) for analysis. Sample solution (1 ml) was mixed
184
with 2.5 ml of sodium phosphate buffer (0.2 M, pH 6.6) and 2.5 ml of 1% (w/v) potassium
185
ferricyanide. The mixture was incubated at 50 °C for 20 min. Then, 2.5 ml of 10%
186
trichloroacetic acid were added. After centrifugating at 1000 × g for 10 min, 2.5 ml of the
187
supernatant were collected and mixed with 2.5 ml of distilled water and 0.4 ml of 0.1% (w/v)
188
ferric chloride in a test tube. After incubation at room temperature for 10 min, the absorbance
189
was measured at 700 nm. Distilled water, instead of the sample, was used as the blank. The
190
reducing power of ascorbic acid was also assayed for comparison. Decreased absorbance, at
191
700 nm, of the reaction mixture indicated decreased reducing capacity. The concentration of
192
the test sample needed to raise the absorbance at 700 nm to 0.5 was evaluated.
193 194 195 196
2.10. ORAC assay The peroxyl radical-scavenging activity of ART was measured according to the method of Lijun You, Mouming Zhao, and Ruihai Liu (2011).
197 198
2.11. HPLC analysis of purified anthocyanin extract
199
HPLC analyses were performed using a Waters 600 pump (Waters, Milford, MA) equipped
200
with a Waters 2998 photodiode array detector at 520 nm. Separation was performed with a
201
Waters C18 column (5 μm, 250 mm × 4.6 mm, i.d.) at 30 °C. Elution was carried out by using
202
a gradient procedure with a mobile phase containing solvent A (0.1% TFA in water) and 9
203
solvent B (acetonitrile) as follows: 0–5 min, 10–15% B; 5–10 min, 15-18% B; 10-20 min,
204
18-20% B; 20–25 min, 20−23% B; 25–30 min, 23-10% B; 40–45 min; 10% B (Paola-Naranjo,
205
Sánchez-Sánchez, & González-Paramás, 2004). The solvent flow rate was 1.0 ml/min, and the
206
injection volume was 20 μl.
207 208
2.12. Statistical analysis
209
The total anthocyanin content was measured in triplicate and data represent mean values ±
210
standard deviation (n = 3). Samples were analyzed in triplicate and one-way analysis of
211
variance performed using SPSS 11.5 (SPSS Inc., Chicago, IL, USA). Significant differences
212
were detected at P <0.05.
213 214
3. Results and discussion
215
3.1. Total anthocyanin content
216
The total anthocyanin content of the purified Rhodomyrtus tomentosa extract, determined
217
by the pH differential method, was 62.8 ± 1.2 mg/100g of freeze-dried weight of R.tomentosa
218
fruits, expressed as cyanidin-3-O-glucoside and reported as the average of three
219
determinations. Cyanidin-3-O-glucoside was the major anthocyanin detected in large amount
220
(47%), followed by peonidin-3-O-glucoside (34%) and malvidin-3-O-glucoside (8%).
221 222
3.2. Antioxidant activity
223
3.2.1. Radical-scavenging activity
224 225
DPPH and ABTS radical-scavenging activities of the purified anthocyanin extracted from R. tomentosa and ascorbic acid control are shown in Table 1 and Fig. 2, respectively.
226
As shown in Fig. 2A, the purified anthocyanin extract showed strong DPPH
227
radical-scavenging activity in a dose-dependent manner, and exhibited good DPPH 10
228
radical-scavenging activity at the concentration of 2 μg/ml and it almost completely inhibited
229
DPPH radicals (> 90%) at a concentration of 20 µg/ml. The IC50 value of DPPH
230
radical-scavenging activity was 6.27 ± 0.25 μg/ml (Table 1). Furthermore, DPPH
231
radical-scavenging activity was linearly correlated (positive) with the concentrations of
232
anthocyanin extracts from 0 to10 μg /ml, while ascorbic acid exhibited the activity from 2 to
233
30 μg/ml, respectively.
234
ABTS•+ is often used for in vitro determination of free radical activity and the relative
235
ability to scavenge the ABTS•+ radical has been compared with the standard trolox. Fig. 2B
236
shows a steady increase in ABTS radical-scavenging capacity, up to a concentration of 0.20
237
mg/ml, that is equivalent to 1.32 mg/ml of trolox. The IC50 value of ABTS radical-scavenging
238
activity was 90.3 ± 1.52 μg/ml.
239
It is noteworthy that both DPPH and ABTS radical-scavenging activities of the tested
240
extract were higher than those of VC (IC50 = 6.27 ± 0.25 μg/ml, 206 ± 2.37 μg/ml) which is
241
always considered as an excellent tool for determining the antioxidant activity of
242
hydrogen-donating antioxidants and of chain breaking antioxidants.
243 244
3.2.2. Reducing power activity
245
Reducing power is often used as an indicator of electron-donating activity, which is an
246
important mechanism for testing antioxidative action of phenolics. The reducing powers of
247
ART and ascorbic acid control are shown in Table 1 and Fig. 2C, respectively. As shown in
248
Fig.2C, the tested ART extract exhibited high reducing power in a dose-dependent manner,
249
but showed a slightly weaker ability compared with those of ascorbic acid. The value, raising
250
the absorbance at 700 nm to 0.5, of reducing power was 51.7 ± 0.74 μg/ml (Table 1).
251
Furthermore, the reducing power was linearly correlated (positive) with the concentrations of
252
anthocyanin extracts and ascorbic acid from 0 to 0.08 mg/ml. The corresponding correlation 11
253
coefficients were 0.996 for anthocyanin extracts (Y = 7.591X + 0.107) and 0.995 for ascorbic
254
acid (Y = 15.17 X + 0.026), respectively.
255 256
3.2.3. ORAC capacity test
257
The ORAC assay is the only antioxidant test that combines both inhibition time and degree
258
of inhibition into a single quantity. The assay uses a biologically relevant radical source, and is
259
also an assay where an added antioxidant competes with a substrate (fluorescein) for the
260
radicals generated by thermal decomposition of azo compounds, like AAPH, and inhibits or
261
retards substrate oxidation (Walton, Lentle, Reynolds, Kruger, & Mcghie, 2006). As
262
mentioned in 2.9, final ORAC values were expressed as μmol of trolox equivalents (TE) /mg
263
of ART, and a higher ORAC value indicated stronger antioxidant activity. As shown in Table
264
1, The ORAC value of the tested ART extract was 9.29 ± 0.08 μmol TE/mg, which was
265
significantly higher than that of ascorbic acid (1.79 ± 0.03 μmolTE/mg). The result indicated
266
that ART exhibited very good antioxidant activity.
267 268
3.3. HPLC analysis
269
Under the optimital HPLC separation condition, a satisfactory separation of the purified
270
anthocyanin extract of R. tomentosa was obtained and the HPLC chromatogram is shown in
271
Figure 3A.
272
The Major anthocyanins represented about 99% of the total peak area with regard to the
273
UV–Vis spectrum taken on-line during HPLC and chromatographic features. However, other
274
minor peaks were also detected which had percentage areas of less than 1%. No UV
275
absorbance maxima in the 310-320 nm range were detected, indicating no acylation of
276
anthocyanins with aromatic acids (Giusti & Jing, 2001).
277 12
278
3.4. Identification of compounds
279
The detailed MS data, including retention times, molecular ion peaks, MS2 fragments and
280
percent area at 520 nm, of all anthocyanins are summarized in Table 2. Fig.4 shows the
281
electrospray mass spectrum and the structures of the isolated anthocyanins.
282
Two major peaks, peak 2 and peak 5 (P2, P5) were obtained as amorphous red powder. As
283
shown in Table 2, P2 and P5 showed peaks at 449 m/z and 463 m/z from ESI–MS, which
284
were in accordance with the mass calculated for C21H21O11 (449.1) and C22H23O11 (463), on
285
the basis of the MS2 detected mass fragments at m/z 287 and m/z 301 related to the loss of one
286
hexose ([M-162]+) molecule, respectively, and the UV–Vis spectrum of the two compounds
287
showed the visible λmax to be 516 nm with the ratio of A440nm/Aλmax exceeding 0.20,
288
corresponding to cyanidin-3-O-glucoside and peonidin-3-O-glucoside (Cerezo et al., 2010;
289
Zhang, Xue, Yang, Ji, & Jiang, 2004). The NMR data of P2 and P5 were as follows:
290
Peak 2 (P2): 1H NMR (CF3COOD-CD3OD): δ6.64 (1H, d, J=1.2 Hz, H-6), δ6.86 (1H, d,
291
J=1.2 Hz, H-8), 8.22 (1H, dd, J = 8.8, 2.2 Hz, H-6´ ), 8.02 (1H, d, J = 2.2 Hz, H-2´ ), 7.04 (1H,
292
d, J = 8.8 Hz, H-5´ ), 8.98 (1H, s, H-4); 5.31 (1H, d, J = 7.8 Hz, H-1 glc), 3.69 (1H, m, H-2
293
glc), 3.56 (2H, m, H-3,5 glc), 3.46 (1H, m, H-4 glc), 3.73 (1H, dd, J = 12.2, 5.6 Hz, H-6b glc),
294
3.96 (1H, dd, J = 12.2, 2.2 Hz, H-6a glc). 13C NMR (CF3COOD-CD3OD): δ164.5 (C-2), 145.8
295
(C-3), 137.2 (C-4), 159.6 (C-5), 103.7 (C-6), 170.8 (C-7), 95.4 (C-8), 157.9 (C-9), 113.6
296
(C-10), 121.5 (C-1´ ), 118.7(C-2´ ), 147.6(C-3´ ), 156.0 (C-4´ ), 117.7 (C-5´ ), 128.4 (C-6´ ),
297
104.1 (C-1 glc), 75.0 (C-2 glc), 78.3 (C-3glc), 71.3 (C-4 glc), 79.0 (C-5 glc), 62.6 (C-6 glc).
298
Peak 5 (P5): 1H NMR ( CF3COOD-CD3OD): δ6.64 (1H, d, J=1.2Hz,,H-6), 6.88 (1H, d,
299
J=1.2 Hz,, H-8), 7.02 (1H, d, J = 8.4 Hz, H-5´ ), 8.21 (1H, dd, J = 8.4, 2.0 Hz, H-6´ ) , 8.16
300
(1H, d, J = 2.0 Hz, H-2´ ), 8.99 (1H, s, H-4), 3.99 (3H, s, OCH3); 5.31 (1H, d, J =8.0 Hz, H-1
301
glc), 3.65 (1H, m, H-2 glc), 3.56 (2H, m, H-3,5 glc), 3.46 (1H, m, H-4 glc), 3.73 (1H, dd, J =
302
12.0, 4.0 Hz, H-6b glc), 3.94 (1H, dd, J = 12.0, 2.0 Hz, H-6a glc). 13
13
C
303
NMR(CF3COOD-CD3OD): δ163.2 (C-2), 144.7 (C-3), 136.5 (C-4), 158.5 (C-5), 103.1 (C-6),
304
170.6 (C-7), 94.5 (C-8), 157.0 (C-9), 112.8 (C-10), 120.3 (C-1´), 114.5 (C-2´), 148.7 (C-3´),
305
155.7 (C-4´), 116.8 (C-5´), 128.1 (C-6´), 103.5 (C-1 glc), 75.1 (C-2 glc), 78.5 (C-3glc), 71.1
306
(C-4 glc), 78.8 (C-5 glc), 62.5 (C-6 glc), 57.0(OCH3).
307
The 1H NMR spectra of P2 showed the presence of two meta-coupled doublet (J = 1.2 Hz)
308
protons on the A-ring at δ 6.64 and 6.86 ppm, which were assigned to H-6 and H-8,
309
respectively. Two sets of doublet and one set of double doublet of an ABM system at δ 7.04
310
(1H, d, J = 8.8 Hz), 8.22 (1H, dd, J = 8.8, 2.2 Hz), 8.02 ppm (1H, d, J = 2.2 Hz) were observed
311
which were characteristic of H-5´, H-6´ and H-2´, respectively. The ring C was a flavanone
312
moiety. The proton signal at δ 3.46-5.31 (H-1-glc~H-6-glc) showed a D-glucopyranose moiety
313
which was assigned as β - configuration based on the large proton-coupling constants of its
314
anomeric proton δ 5.31 ppm (1H, d, J = 7.8 Hz, H-1´) (Byamukama, Kiremire, Andersen, &
315
Steigen, 2005). The cross peak at δ5.31/145.8 (H-1glc/C-3) in the HMBC spectrum of P2
316
indicated that the sugar moiety was attached to C-3, which was confirmed by Zarena and
317
Sankar (2012). Based on the above results and literature (Lee & ChoungL, 2011), P2 was
318
identified as cyanidin-3-O-glucopyranoside.
319
Comparing the 1H and 13C NMR spectra of P5 with those of P2, its spectral features were
320
closely similar to those of P2, except for the excess of the three-proton signal at δ 3.99 (3H, s)
321
in the 1H NMR spectra and one carbon signal at δ 57.0 in the
322
due to a methyl group attached to oxygen. Further support for this structure was obtained by
323
the mass spectrum of P5 displaying a [M]+ ion peak at m/z 463, corresponding to the excess of
324
an methylene moiety (14 mass units) from P2 ([M]+). Based on the above evidence, the
325
structure of P5, which differed from P2 by a methyl group attached to the H-3´ position
326
instead of a hydroxyl group attached to the H-3´ position on the B ring entity, was deduced to
327
be peonidin-3-O-glucoside and the chemical structure of P2 was further confirmed by 14
13
C NMR spectra, which was
328
comparison of the NMR and MS data with the literature (Fossen, Slimestad, Øvstedal, &
329
Andersen, 2002).
330
Peak 1 (P1): amorphous red powder; ESI-MS m/z 465; 1H NMR (CF3COOD-CD3OD):
331
δ6.66 (1H, d, J=1.2 Hz, H-6), 6.87 (1H, d, J=1.2 Hz, H-8), 7.74 (2H, d, J = 2.2 Hz, H-2´, 6´ ),
332
8.93 (1H, s, H-4); 5.34 (1H, d, J = 7.8 Hz, H-1 glc), 3.73 (1H, m, H-2 glc), 3.60 (2H, m, H-3,5
333
glc), 3.51 (1H, m, H-4 glc), 3.79 (1H, dd, J = 12.2, 5.4 Hz, H-6b glc), 3.94 (1H, dd, J = 12.2,
334
2.0 Hz, H-6a glc). 13C NMR(CF3COOD-CD3OD): δ164.2 (C-2), 144.6 (C-3), 136.8 (C-4),
335
159.4 (C-5), 103.1 (C-6), 170.8 (C-7), 95.4 (C-8), 157.8 (C-9), 113.0 (C-10), 121.5 (C-1´ ),
336
113.7(C-2´ ), 147.6(C-3´ ), 146.0 (C-4´ ), 147.9 (C-5´ ), 113.4 (C-6´ ), 103.5 (C-1 glc), 74.8
337
(C-2 glc), 78.0 (C-3 glc), 71.2 (C-4 glc), 78.8 (C-5 glc), 62.3 (C-6 glc).
338
The MS analysis of P1 (tR =11.75 min) showed an [M]+ ion at m/z 465 and a major
339
fragmentation in MS2 at m/z 303 (-162 amu) which would correspond to the monoglucoside of
340
delphinidin (Cerezo et al., 2010), The UV–Vis spectrum of this compound showed the visible
341
λmax to be 523 nm; the ratio of absorbance at 440nm to the absorbance at visible maximum
342
wavelength (A440nm/Aλmax ratio) for peak 1 was found to be 0.28, indicating that the compound
343
is delphinidin-3-O-glucoside (Longo & Vasapollo, 2006).
344
The 1H NMR spectra of P1 showed the presence of two meta-coupled doublet (J = 1.2 Hz)
345
protons on the A-ring at δ 6.66 and 6.87 ppm, which were assigned to H-6 and H-8,
346
respectively. One set of doublets of an AM system at δ 7.74 (2H, d, J = 2.2 Hz) was observed
347
which was characteristic of H-2´ and 6´.The analysis of the NMR spectral data also revealed
348
only a single glucose moiety with a proton signal at δ5.34 (1H, d, J = 7.8 Hz) coupled with the
349
C-3 aglycone at 144.6 ppm, indicating a D-glucopyranose moiety with a β-configuration
350
attached to the C-3 position. Thus the structure of P1 was elucidated to be
351
delphinidin-3-O-glucoside and was in agreement with the report of Pazmiño-Durán, Giusti,
352
Wrolstad and Glória (2001). 15
353
Peak 3 (P3): amorphous purple powder; ESI-MS m/z 479; 1H NMR (CF3COOD-CD3OD):
354
δ6.64 (1H, d, J=1.2 Hz, H-6), 6.89 (1H, d, J=1.2 Hz, H-8), 7.74 (1H, d, J = 2.2 Hz, H-6´ ),
355
7.90 (1H, d, J = 2.2 Hz, H-2´ ), 8.97 (1H, s, H-4), 3.99 (3H, s, OCH3); 5.33 (1H, d, J = 8.0 Hz,
356
H-1 glc), 3.69 (1H, dd, m, H-2 glc), 3.57 (2H, m, H-3,5 glc), 3.42 (1H, m, H-4 glc), 3.74 (1H,
357
dd, J = 12.2, 5.6 Hz, H-6b glc), 3.92 (1H, dd, J = 12.2, 2.0 Hz, H-6a glc).
358
NMR(CF3COOD-CD3OD): δ162.9 (C-2), 145.0 (C-3), 135.6 (C-4), 158.9 (C-5), 103.4 (C-6),
359
170.6 (C-7), 95.5 (C-8), 157.1 (C-9), 113.3 (C-10), 119.7 (C-1´ ), 109.6 (C-2´ ), 149.5 (C-3´ ),
360
146.0 (C-4´ ), 147.7 (C-5´ ), 113.0(C-6´ ), 103.5 (C-1 glc), 74.5 (C-2 glc), 78.0 (C-3glc), 71.0
361
(C-4 glc), 78.2 (C-5 glc), 62.6 (C-6 glc), 57.5( OCH3).
13
C
362
The ESI-MS spectrum of peak 3(tR = 13.73 min) was characterized by an ion signal at m/z
363
479 with an MS2 fragment at m/z 317 ([M-162]+) coinciding with the molecular formula
364
C22H23O12 of petunidin aglycone linked with a glucose moiety (Lin, Harnly, Pastor-Corrales,
365
& Luthria, 2008). The UV–Vis spectrum of pigment 3, taken on-line during HPLC, showed a
366
visible maximum at 526 nm.
367
Comparing the 1H and 13C NMR spectra of P3 with those of P1, its spectral features were
368
closely similar to those of P1, except for the excess of the three-proton signal at δ 3.99 (3H, s)
369
in 1H NMR spectra and one carbon signal at δ 57.5 in 13C NMR spectra due to a methyl group
370
attached to the H-3´ position instead of a hydroxyl group attached to the H-3´ position on the
371
B ring entity. By comparison with previous research data (Lee et al, 2009), P3 was assigned as
372
petunidin-3-O-glucoside.
373
Peak 4 (P4): amorphous red powder; ESI-MS m/z 433; 1H NMR (CF3COOD-CD3OD): δ
374
6.67 (1H, d, J =1.2 Hz, H-6), 6.94 (1H, d, J = 1.2 Hz, H-8), 7.05 (2H, d, J = 8.8 Hz, H-3´, 5´ ),
375
8.60 (2H, d, J = 8.8 Hz, H-2´, 6´ ), 9.08 (1H, s, H-4), 5.34(1H, d, J = 8.0 Hz, H-1 glc), 3.68
376
(1H, m, H-2 glc), 3.54 (2H, m, H-3,5 glc), 3.42 (1H, m, H-4 glc), 3.80 (1H, dd, J =12.2, 6.0
377
Hz, H-6b glc), 4.04 (1H, dd, J = 12.2, 2.0 Hz, H-6a glc). 13C NMR (CF3COOD-CD3OD): 16
378
δ164.8 (C-2), 145.8 (C-3), 137.4 (C-4), 159.9 (C-5), 103.7 (C-6), 170.9 (C-7), 95.5 (C-8),
379
158.1 (C-9), 114.5 (C-10), 121.5 (C-1´ ), 136.8 (C-2´ ), 118.6 (C-3´ ), 166.0 (C-4´ ), 118.7
380
(C-5´ ), 136.4 (C-6´ ), 104.1 (C-1 glc), 75.3 (C-2 glc), 78.5 (C-3glc), 71.3 (C-4 glc), 79.0 (C-5
381
glc), 62.6 (C-6 glc).
382
The molecular ion [M]+ at m/z 433 received from the ESI-MS analysis of P4 (tR =15.46 min)
383
confirmed the molecular formula, C21H21O10, for glucoside derivatives of pelargonidin
384
aglycone. The fragment ion [M+H-162]+ at m/z 271 was consistent with the structure of
385
pelargonidin with a loss of glucose moiety from pelargonidin-3-O-glucoside (Hong &
386
Wrolstad, 1990). The UV–Vis spectrum of this compound showed the visible λmax at 506 nm.
387
The 1H NMR and
13
CNMR spectra suggested that P4 contained a flavanone moiety; the
388
proton signals at δ 6.67(1H, d, J =1.2 Hz) and 6.94 (1H, d, J = 1.2 Hz) implied the presence of
389
two meta-coupled doublet protons on the A-ring which were assigned to H-6 and H-8,
390
respectively. Two sets of doublets of an AB system at δ 7.05 (2H, d, J = 8.8 Hz) and 8.60 (2H,
391
d, J = 8.8 Hz) were observed, which were characteristic of H-3´, H-5´ and H-2´, H-6´,
392
respectively. As P2, the large coupling constants (J = 8 Hz) for the anomeric protons (δ5.34,
393
1H, d, J = 8.0 Hz, H-1 glc) confirmed the presence of a β-D-glucosidic linkage in P4. By
394
comparison with the literature, P4 was further confirmed as pelargonidin-3-O-glucoside
395
(Pedersen, Andersen, Aksnes, & Nerdal, 1993).
396
Peak 6(P6): amorphous dark red powder; ESI-MS m/z 493; 1H NMR (CF3COOD-CD3OD):
397
δ6.74(1H, d, J = 1.9 Hz, H-6), 7.06 (1H, d, J = 1.9 Hz, H-8), 7.96 (2H, d, J = 2.2 Hz, H-2´, 6´ ),
398
8.96(1H, s, H-4), 3.90 (6H, s, 2×CH3); 5.37 (1H, d, J = 7.8 Hz, H-1 glc), 3.64(1H, m, H-2 glc),
399
3.74 (1H, m, H-3 glc), 3.79 (1H, m, H-5 glc), 3.43 (1H, m, H-4 glc), 3.51 (1H, dd, J = 12.4,
400
4.8 Hz, H-6b glc), 3.96 (1H, dd, J = 12.4, 2.2 Hz, H-6a glc). 13C NMR (CF3COOD-CD3OD):
401
δ163.0 (C-2), 145.0 (C-3), 136.2 (C-4), 158.9 (C-5), 103.1 (C-6), 170.5 (C-7), 95.1 (C-8),
402
157.4 (C-9),112.9 (C-10), 120.0 (C-1´ ), 110.5 (C-2´ ), 149.1 (C-3´ ), 146.7 (C-4´ ), 149.2 17
403
(C-5´ ), 111.0(C-6´ ), 103.8 (C-1 glc), 74.6 (C-2 glc), 78.0 (C-3glc), 70.9 (C-4 glc), 77.8 (C-5
404
glc), 67.3 (C-6 glc), 57.3(2×OCH3).
405
The last peak in the chromatogram was peak 6, with the MS and MS2 profiles from ESI-MS
406
spectra showing strong ion peaks at m/z 493 and 331 for peak 6 (tR = 15.91 min) coinciding
407
with the molecular ion of malvidin-3-O-glucoside with a loss of a glucose moiety (Li, Wang,
408
Guo, & Wang, 2011). Based on the spectrum data and comparing with previously reported
409
data (Alcalde-Eon, Escribano-Bailón, Santos-Buelga, & Rivas-Gonzalo, 2006), P6 was
410
identified as malvidin-3-O-glucoside.
411
As shown in Figure 3A, there are six principal anthocyanin peaks, and two major peaks
412
(peak 2 and peak 5) with concentrations of 47% (29.4 ± 0.39 mg/100g) and 34 % (21.3 ± 0.33
413
mg/100g) of the total peak area with retention times of 13.20 and 15.46 min, respectively,
414
(Table 2). The elution times of the other four peaks with the concentration of 3.35% (peak 1,
415
2.07 ± 0.03 mg/100g), 5.42% (peak 3, 3.51 ± 0.14 mg/100g), 0.85% (peak 4, 0.49 ± 0.05
416
mg/100g), 8.86% (peak 6, 5.69 ± 0.16 mg/100 g) were 11.75 (peak 1), 13.73 (peak 3), 14.78
417
(peak 4), 15.90 (peak 6) min. The final structures of the six compounds isolated and identified
418
are shown in Figure 3B.
419
Based on the available literature, there have been only a few reports in previous studies
420
regarding the extraction and preliminary qualitative research of the anthocyanins in R.
421
tomentosa fruits. This is the first time that all the major anthocyanins were systematically
422
isolated and identified from R.tomentosa fruits.
423 424
4. Conclusions
425
The total anthocyanin content of the purified Rhodomyrtus tomentosa (Ait.) Hassk fruits
426
extract was 62.8 ± 1.2 mg/100 g of freeze-dried weight of R.tomentosa fruits and it possessed
427
excellent in vitro free radical-scavenging activity. Pure anthocyanins were isolated and 18
428
analyzed by ESI-MS and NMR spectroscopy. The six anthocyanin structures characterised
429
were
430
pelargonidin-3-O-glucoside, peonidin-3-O-glucoside, and malvidin-3-O-glucoside. As a result,
431
this study has systematically documented, for the first time, the presence of six anthocyanin
432
derivatives from the tested extract. Fruits of R. tomentosa also contain important amounts of
433
other phenolics, amino acids and ascorbic acid. These phytochemicals may partially explain
434
the diverse bioactive properties of this plant. Therefore, R. tomentosa could be a source of
435
functional substances for human health and the food industry. Further investigation is to
436
compare the differences of the type and content of anthocyanins in Rhodomyrtus tomentosa of
437
different regions in China to provide a theoretical support for further development of this
438
resource.
delphinidin-3-O-glucoside,
cyanidin-3-O-glucoside,
petunidin-3-O-glucoside,
439 440
Acknowledgements
441
The authors are grateful to the National Natural Science Foundation of China (No.
442
31201416, 31000759 and 31101222) and Technology Program (Nos. 2011BAD23B01) for
443
their financial support.
444 445
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446
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Zarena, A. S., & Sankar, K. U. (2012). Isolation and identification of pelargonidin 3- glucoside
525
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526
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527
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528
Figure captions:
529
Fig. 1 The fruit and flower of Rhodomyrtus tomentosa (Ait.) Hassk collected in Guangdong
530
province.
531
Fig. 2 Antioxidant activities of ART extracted from Rhodomyrtus tomentosa fruit determined
532
by DPPH and ABTS free radical-scavenging assays (A and B) and reducing power assay(C).
533
Fig. 3 HPLC chromatogram (A) showing the six anthocyanin profiles of Rhodomyrtus
534
tomentosa extract monitored at 520 nm. (Table 2 shows the peak identification) and the
535
structures of the anthocyanins (B) isolated from the fruits of Rhodomyrtus tomentosa.
536
Fig. 4 Electrospray mass spectrum of identified anthocyanins. Peak 1:
537
delphinidin-3-O-glucoside; Peak 2: cyanidin-3-O-glucoside; Peak 3: petunidin-3-O-glucoside;
538 539
Peak 4: pelargonidin-3-O-glucoside; Peak 5: peonidin-3-O-glucoside; Peak 6: malvidin-3-O-glucoside.
23
540
Table 1 Antioxidant activities of anthocyanins (ART) extracted from Rhodomyrtus tomentosa
541
and ascorbic acid using the DPPH• assay, ABTS+• assay, reducing power assay and ORAC test IC50 / DPPH•
IC50 / TEAC
A700nm=0.5/Reducing
ORAC value
(μg/ml)1
(μg/ml)2
Power(μg/ml)3
(μmolTE /mg)
ART
6.27 ± 0.25b
90.3 ± 1.52b
51.7 ± 0.74a
9.29 ± 0.08a
ascorbic acid
17.4 ± 0.31a
206 ± 2.37a
31.3 ± 0.93b
1.79 ± 0.03b
Samples
542
All the trials were performed in triplicate (n = 3) and all the data represent the means ±
543
standard deviation (n > 3). Data in the same column with different letters are significantly
544
different (p < 0.05).
545
1
546
needed to decrease the absorbance at 517nm by 50%.
547
2
548
decrease the absorbance at 734nm by 50%.
549
3
550
the absorbance at700nm to 0.5.
The antioxidant activity was calculated as the concentration of the test sample
The antioxidant activity was evaluated as the concentration of the test sample required to
The antioxidant activity was evaluated as the concentration of the test sample needed to raise
24
551
Table 2 LC-MS characteristics of anthocyanins separated from Rhodomyrtus tomentosa: retention time, wavelengths of maximum absorption
552
(λmax), molecular ion, fragmentation pattern and tentative identification
553
a, b
The fragment ions are shown in order of their relative abundance.
Peak.No.
Elution
Peak
λmax
Molecular
Mass loss
MS2 of
Peak
Contents
time(min)
Area(%)
(nm)
ion[M+]，m/za
(M+H+)-MS2
[M+]，m/zb
assignment
(mg/100g
(520nm))
m/z
dry fruits)
1
11.75
3.35
523/277
465
-162
302.9
Delphinidin-3-O-glucoside
2.07 ± 0.03
2
13.20
47.27
516/280
449/286.9
-162
286.9
Cyanidin-3-O-glucoside
29.4 ± 0.39
3
13.73
5.42
526/277
479/316.9
-162
316.9
Petunidin-3-O-glucoside
3.51 ± 0.14
4
14.78
0.85
516/281
433/271.0
-162
271.0,415.1
Pelargonidin-3-O-glucoside
0.49 ± 0.05
5
15.46
34.22
506/279
463/300.9
-162
300.9
Peonidin-3-O-glucoside
21.3 ± 0.33
6
15.90
8.86
527/277
493/331.0
-162
331.0
Malvidin-3-O-glucoside
5.69 ± 0.16
25
554
555
Fig. 1
26
DPPH•-Scavenging Capacity (%)
556
A 100 80 60 ART Vc
40 20 0 0
4
8
12
16
Concentration
557
mg trolox equivalent /mg
B
20
24
28
32
0.18
0.21
( µ g/ml )
1.4 1.2
ART Vc
1.0 0.8 0.6 0.4 0.2 0.0 0.00
0.03
0.06
0.09
0.12
Concentration(mg/ml)
27
0.15
Reducing Power A700 nm
C
558
2.0 ART Vc
1.6 1.2 0.8 0.4 0.0 0.00
0.02
0.04
0.06
Concentration(mg/ml)
559
Fig. 2
28
0.08
0.10
560
561
Fig. 3
29
562
563
Fig. 4
30
564
Research Highlights
565
1. All the major anthocyanins were isolated and identified from R.tomentosa fruits.
566
2. Cyanidin-3-O-glucoside was considered as the most abundant antocyanin.
567
3. The purified anthocyanin extract showed strong antioxidant activities.
568 569
31
S0308-8146(13)00142-8 http://dx.doi.org/10.1016/j.foodchem.2013.01.107 FOCH 13656
To appear in:
Food Chemistry
Received Date: Revised Date: Accepted Date:
30 October 2012 5 January 2013 28 January 2013
Please cite this article as: Cui, C., Zhang, S., You, L., Ren, J., Luo, W., Chen, W., Zhao, M., Antioxidant capacity of anthocyanins from Rhodomyrtus tomentosa (Ait.) and identification of the major anthocyanins, Food Chemistry (2013), doi: http://dx.doi.org/10.1016/j.foodchem.2013.01.107
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1
Antioxidant capacity of anthocyanins from Rhodomyrtus tomentosa (Ait.) and
2
identification of the major anthocyanins
3
Chun Cui1, Shaomin Zhang1, Lijun You1, Jiaoyan Ren1, Wei Luo2, Wenfen Chen1, Mouming
4
Zhao1,*
5
1
College of Light Industry and Food, South China University of Technology, Guangzhou
6 7
510640, China 2
Analysis and Testing Center, South China University of Technology, Guangzhou 510640,
8
China
9 10 11
Corresponding author
12
Mouming Zhao, Professor
13
Tel/Fax: +86 20 87113914
14
E-mail: [email protected]
1
15
ABSTRACT
16
The anthocyanins in the fruits of Rhodomyrtus tomentosa (ACN) were extracted by 1%
17
TFA in methanol, and then purified by X-5 resin column and C18 (SPE) cartridges. The
18
purified anthocyanin extract (ART) from the fruits of Rhodomyrtus tomentosa showed strong
19
antioxidant activities, including DPPH radical-scavenging capacity, ABTS radical scavenging
20
capacity, reducing power and oxygen radical absorbance capacity (ORAC). The purified
21
anthocyanin extract was analyzed by high performance liquid chromatography (HPLC). The
22
major anthocyanins were purified by semi-preparative HPLC and Sephadex LH-20 column
23
chromatography, and were identified as cyanidin-3-O-glucoside, peonidin-3-O-glucoside,
24
malvidin-3-O-glucoside,
25
pelargonidin-3-glucoside by HPLC-ESI/MS and nuclear magnetic resonance spectroscopy
26
(NMR). Cyanidin-3-O-glucoside was considered as the most abundant anthocyanin, which
27
was 29.4 mg/100 g dry weight of R. tomentosa fruits. Additionally, all the major anthocyanins
28
were identified from R. tomentosa fruit for the first time.
29 30
Keywords:
petunidin-3-O-glucoside,
Rhodomyrtus
tomentosa;
Cyanidin-3-O-glucoside
2
delphinidin-3-O-glucoside
Anthocyanins;
Antioxidant
and
activity;
31
1. Introduction
32
Rhoddmyrtus tomentosa (Ait.) Hassk, a member of the Myrtaceae family, commonly known
33
as rose myrtle, is an abundant evergreen shrub native to southeast Asia, with rose-pink flowers
34
and dark-purple edible bell-shaped fruits (Amporn, Tony, & John, 2005). The stem, leaf, fruits
35
of the whole plant can be used as medical materials. The R. tomentosa fruit possesses
36
excellent pharmacological properties, including antibacterial activity against Gram-positive
37
bacteria, such as Streptococcus pyogenes and Escherichia coli (Dachriyanus et al., 2002;
38
Surasak & Supayang, 2008).
39
Rhodomyrtus tomentosa fruit is widely distributed in south China; its bright purplish-red
40
colour is due to anthocyanins. Anthocyanins, an important group of water-soluble pigments in
41
natural products, are widely spread in flowers, fruits and leaves. They usually link with sugar
42
moieties and constitute flavonoids, attracting more and more attention due to their usage as
43
natural food additives and excellent functional properties for human health (Kaliora,
44
Dedoussis, & Schmidt, 2006; Li, Wang, Guo, & Wang, 2011). On the basis of their structural
45
characteristics, anthocyanins possess various biological activities, including antioxidant
46
(Cerezo, Cuevas, Winterhalter, Garcia-Parrilla, & Troncoso, 2010), anticancer (Wang & Stoner,
47
2008), anti-inflammatory (Greenspan, Bauer, Pollock, Gangemi, Mayer, & Ghaffar, 2005),
48
anti-artery atherosclerosis, anti-hypertensive (Pinent, Blay, Bladé, Salvadó, Arola, & Ardévol,
49
2004) and antibacterial activities (Lacombe, Wu, Tyler, & Edwards, 2010). In recent decades,
50
the antioxidant activities of anthocyanin and its working mechanism have attracted growing
51
global interest. As reported, anthocyanin might play its protective role through the working
52
system of H atom transfer, single electron transfer and metal chelation (Monica, Nino, &
53
Marirosa, 2011). However, to our knowledge, the information regarding the antioxidant
54
capacity and major anthocyanins of Rhodomyrtus tomentosa is limited.
55
The objectives of the present study were to extract and purify the anthocyanins from the 3
56
fruit of R. tomentosa and to evaluate their antioxidant capacity. The major anthocyanins were
57
further isolated by semi-preparative HPLC and column chromatography, and identified by
58
HPLC-ESI-MS and NMR spectroscopy.
59
2. Materials and methods
60
2.1. Plant material
61
The wild-grown mature fruits of Rhodomyrtus tomentosa (Ait.) Hassk were collected in
62
Shanwei, Guangdong Province, China, in August (Fig. 1), freeze-dried after being washed
63
with clean sterile water, and then stored at -20oC prior to extraction.
64 65
2.2. Chemicals
66
2,2´-Azobis
(2-methylpropionamidine)
dihydrochloride
(AAPH),
2,2-diphenyl-1-
67
picrylhydrazyl (DPPH), 2,2´-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid) (ABTS),
68
6-hydroxy-2,5,7,8-tetramethyl-2-chromanecarboxylic acid (trolox), sodium fluorescein, 3´,6´-
69
dihydroxyspiro [isobenzofuran-1[3H],9´[9H]-xanthen] -3-one(FL), ascorbic acid, CF3COOD
70
and CD3OD were purchased from Sigma Chemical Co. (St. Louis, MO,USA). X-5 resins were
71
obtained from Haiguang Chemical Co. Ltd., (Tianjin, China). Sephadex LH-20 was purchased
72
from Pharmacia Fine Chemicals Co. (Uppsala, Sweden) and Sep-Pak cartridges were
73
purchased from Waters Co., (Milford, Bedford, MA, USA). All the solvents for HPLC
74
analysis were of HPLC grade. All the other chemicals used were of analytical grade.
75 76
2.3. Extraction of anthocyanins
77
Free-dried sample (300 g) was macerated with 1000 ml of TFA (trifluoroacetic acid):
78
methanol (1:99; v/v) for 48 h in the dark at room temperature, and the remaining residues were
79
extracted by 400 ml of the extract solvent to extract anthocyanin. The supernatants were
80
obtained by centrifugation (8,000 × g, 15 min) in a GL-21M refrigerated centrifuge (Xiangyi 4
81
Instrument Co. Ltd., Changsha, China) and filtration. Finally, the acidic methanol extracts
82
were combined and evaporated, using a rotary evaporator (RE-52AA, Yarong Instrument
83
Factory, Shanghai, China) at 40 °C.
84 85 86
2.4. Purification of anthocyanins The concentrated crude extract was purified by partition (several times) against ethylacetate
87
and chloroform to remove non-polar compounds.
88
time) phase was then subjected to a X-5 resin column (1.6 × 40 cm) to remove free sugars,
89
aliphatic acids and other water-soluble compounds by washing (several times) with the
90
column volume of distilled water. The adsorbed anthocyanins were eluted using methanol
91
containing 0.1% trifluoroacetic acid (TFA, v/v).
The partial purified aqueous (10 ml each
92
The concentrated anthocyanin extract was further refined by solid phase extraction (SPE) in
93
C18 cartridges (Sep pak, Waters). The aqueous extract of anthocyanin was passed through a
94
sorbent C-18 Sep-Pak cartridge (Waters Associates, Milford, MA) previously activated with
95
acidified methanol (0.01% HCl v/v) and equilibrated with water. The extract adsorbed onto
96
the cartridge was rinsed with ultra-pure water to remove water-soluble impurities, and then
97
eluted with acidified methanol (0.01% HCl, v/v). The acidified methanol solution was
98
evaporated under vacuum, redissolved in water, and lyophilized (R2L-100KPS, Kyowa
99
Vacuum Engineering, Tokyo, Japan). The dry fraction was dissolved with deionized water.
100
Samples were filtered through a 0.45 μm filter before analysis.
101 102
2.5. Isolation and identification of the main anthocyanins from the purified extract
103
2.5.1. Isolation
104
The purified extract, containing the major anthocyanin-derived pigments, was isolated by
105
semi-preparative HPLC, using a Waters X-bridge reversed-phase C18 column (5μm, 10 × 150 5
106
mm, i.d.) at 35 °C with a flow rate of 4.5 ml/min and was monitored at 520 nm. The solvents
107
were (A), water/formic acid (98:2), and (B), formic acid/methanol (2:98), with the following
108
gradient: 10% to 15% B over 5 min, 15% to 18% B over 5min, 18% to 23% B over 20 min,
109
from 23% to 25% B over 10 min. and then each fraction was further chromatographed on a
110
Sephadex
111
methanol/water/trifluoroacetic acid at a ratio of 20:79.5:0.5 to obtain compounds 1 to 6 ,
112
respectively.
LH-20
column
(1.0
×
60
cm),
eluting
with
a
mixture
of
113 114
2.5.2. Molecular weight determination
115
A MS system (Esquire HCT PLUS, Phenomenex, Torrance, CA, USA), equipped with a
116
Hewlett–Packard1100 series liquid chromatography system, was used to determine the
117
molecular weight of each compound. One hundred microlitres of sample solution (100 μg/ml)
118
was injected into the MS system. Mass spectra in the positive-ion mode were generated under
119
the following conditions: HV capillary = 2800 V; HV end plate offset = -500V; nebulizer
120
pressure = 10 psi; dry temperature = 300°C; Dry Gas = 5.00 l/min; m/z range = 100–1000. A
121
reversed-phase column (250 × 4.6 mm, 5 μm, C18), thermostatted at 35 °C, was used for
122
separation; solvents were (A) aqueous 0.1% trifluoroacetic acid, and (B) 100% acetonitrile,
123
establishing the gradient as described in 2.10.
124 125 126
2.5.3. NMR identification The NMR results were obtained at 400 MHz and 100 MHz for 1H and 13C, respectively, on
127
a Bruker AVANCE Spectrometer (Bruker DRX400, Bruker Biospin Co., Karlsruhe, Germany)
128
in the solvent, CF3COOD-CD3OD (5:95; v/v); coupling constants were expressed in Hertz,
129
and chemical shifts were given on a δ (ppm) scale with TMS (tetramethylsilane) or solvent
130
signals as an internal standard. 6
131 132
2.6. Determination of total anthocyanin content (TAC)
133
TAC was determined according to the pH differential method by Kim, Jeong and Lee
134
(2003). Absorbance was measured at 520 and 700 nm in buffers at pH 1.0 and 4.5, using A =
135
[(A520 - A700)pH 1.0 - (A520 - A700)pH 4.5] and expressed as mg of cyanidin-3-glycoside (molar
136
extinction coefficient of 26900 and molecular weight of 449.2) equivalents per 100 g of dry
137
fruit weight. TAC was calculated using the following equation. Data were reported as means ±
138
standard deviation of triplicate determinations.
139
TAC (mg /100g) = A × MW × DF ×
1 V × ×100 ε×L M
(1)
140
where A is absorbance, ε is cyanidin-3-O-glucoside molar absorbance (26900), L is the cell
141
pathlength (1 cm), MW is the molecular weight of cyanidin-3-glucoside (449.2 Da), DF is the
142
dilution factor, V is the final volume (ml), and M is the dry weight (mg).
143 144
2.7. DPPH radical-scavenging activity assay
145
DPPH radical-scavenging activity was measured according to the method of Wu, Chen, and
146
Shiau (2003) with a slight modification. Aliquots (2.0 ml) of 0 (control), 2, 4, 6, 8, 10, 12, 20
147
and 30 μg/ml of anthocyanin extract (ART) dissolved in distilled water were added to 2.0 ml
148
of 0.2 mM DPPH• that was dissolved in 95% ethanol. The mixture was then shaken
149
vigorously using a mixer (QT-1 Mixer, Tianchen Technological Co.Ltd., Shanghai, China).
150
The reaction mixture was incubated for 30 min at 30°C in the dark. The absorbance of the
151
resulting solution was recorded at 517 nm by a spectrophotometer (UV2100, Unico Instrument
152
Co., Ltd., Shanghai, China). The scavenging activity was calculated using the following
153
equation:
154
Scavenging
activity
(%)
＝
(ADPPH•
sample
155
－ Asample
control)
×
100/ADPPH•
blank
(2 7
156
)
157
where ADPPH• sample = absorance of 2 ml of sample solution + DPPH solution; Asample control =
158
absorance of 2 ml of sample solution + 2 ml of 95% ethanol; and ADPPH• blank = absorance of 2
159
ml of 95% ethanol + DPPH• solution. Ascorbic acid was used as the reference. IC50 value (μg
160
compound ml-1), the concentration of extract that was required to scavenge 50% of radicals,
161
was calculated.
162 163
+ 2.8. ABTS • radical cation-scavenging activity assay
164
ABTS radical cation-scavenging activity of anthocyanins was determined as described by
165
+ Wang and Xiong (2005) with a slight modification. The ABTS• solution was prepared with
166
+ final concentrations of 7 mM ABTS• and 2.45 mM potassium persulfate. The solution was
167
incubated for 16 h at room temperature in the dark until the reaction was completed. Prior to
168
+ the assay, the absorbance of the ABTS• solution at 734 nm was adjusted to 0.70 ± 0.02 by
169
dilution with 0.2 M sodium phosphate-buffered saline (pH 7.4). Then 40 μl of ART (30, 50,
170
+ 80, 100, 120, 160, 200 μg/ml) were added to 4 ml of diluted ABTS• solution. The mixture
171
was shaken vigorously for 30 s and allowed to stand in the dark for 6 min. An equivalent
172
volume of distilled water, instead of the sample, was used for the blank. The absorbance of the
173
resultant solution was measured at 734 nm. A standard curve was obtained by using trolox
174
standard solution at various concentrations (0.4, 0.8, 1.2, 1.6, 2.0, 2.4 mM) with ethanol. The
175
standard curve was prepared by reacting 40 μl of trolox (0.4, 0.8, 1.2, 1.6, 2.0, 2.4 mM) with 4
176
+ ml of diluted ABTS• solution. The degree of ABTS radical-scavenging activity of
177
anthocyanins was calculated, based on the trolox standard curve, and was expressed in terms 8
178
of mg trolox equivalents (TE) /mg anthocyanin.
179 180
2.9. Reducing power assay
181
The reducing power of anthocyanins was determined according to the method of Oyaizu
182
(1988) with a slight modification. ART were dissolved in distilled water to obtain various
183
concentrations (10, 20, 40, 60, 80, 100 μg/ml) for analysis. Sample solution (1 ml) was mixed
184
with 2.5 ml of sodium phosphate buffer (0.2 M, pH 6.6) and 2.5 ml of 1% (w/v) potassium
185
ferricyanide. The mixture was incubated at 50 °C for 20 min. Then, 2.5 ml of 10%
186
trichloroacetic acid were added. After centrifugating at 1000 × g for 10 min, 2.5 ml of the
187
supernatant were collected and mixed with 2.5 ml of distilled water and 0.4 ml of 0.1% (w/v)
188
ferric chloride in a test tube. After incubation at room temperature for 10 min, the absorbance
189
was measured at 700 nm. Distilled water, instead of the sample, was used as the blank. The
190
reducing power of ascorbic acid was also assayed for comparison. Decreased absorbance, at
191
700 nm, of the reaction mixture indicated decreased reducing capacity. The concentration of
192
the test sample needed to raise the absorbance at 700 nm to 0.5 was evaluated.
193 194 195 196
2.10. ORAC assay The peroxyl radical-scavenging activity of ART was measured according to the method of Lijun You, Mouming Zhao, and Ruihai Liu (2011).
197 198
2.11. HPLC analysis of purified anthocyanin extract
199
HPLC analyses were performed using a Waters 600 pump (Waters, Milford, MA) equipped
200
with a Waters 2998 photodiode array detector at 520 nm. Separation was performed with a
201
Waters C18 column (5 μm, 250 mm × 4.6 mm, i.d.) at 30 °C. Elution was carried out by using
202
a gradient procedure with a mobile phase containing solvent A (0.1% TFA in water) and 9
203
solvent B (acetonitrile) as follows: 0–5 min, 10–15% B; 5–10 min, 15-18% B; 10-20 min,
204
18-20% B; 20–25 min, 20−23% B; 25–30 min, 23-10% B; 40–45 min; 10% B (Paola-Naranjo,
205
Sánchez-Sánchez, & González-Paramás, 2004). The solvent flow rate was 1.0 ml/min, and the
206
injection volume was 20 μl.
207 208
2.12. Statistical analysis
209
The total anthocyanin content was measured in triplicate and data represent mean values ±
210
standard deviation (n = 3). Samples were analyzed in triplicate and one-way analysis of
211
variance performed using SPSS 11.5 (SPSS Inc., Chicago, IL, USA). Significant differences
212
were detected at P <0.05.
213 214
3. Results and discussion
215
3.1. Total anthocyanin content
216
The total anthocyanin content of the purified Rhodomyrtus tomentosa extract, determined
217
by the pH differential method, was 62.8 ± 1.2 mg/100g of freeze-dried weight of R.tomentosa
218
fruits, expressed as cyanidin-3-O-glucoside and reported as the average of three
219
determinations. Cyanidin-3-O-glucoside was the major anthocyanin detected in large amount
220
(47%), followed by peonidin-3-O-glucoside (34%) and malvidin-3-O-glucoside (8%).
221 222
3.2. Antioxidant activity
223
3.2.1. Radical-scavenging activity
224 225
DPPH and ABTS radical-scavenging activities of the purified anthocyanin extracted from R. tomentosa and ascorbic acid control are shown in Table 1 and Fig. 2, respectively.
226
As shown in Fig. 2A, the purified anthocyanin extract showed strong DPPH
227
radical-scavenging activity in a dose-dependent manner, and exhibited good DPPH 10
228
radical-scavenging activity at the concentration of 2 μg/ml and it almost completely inhibited
229
DPPH radicals (> 90%) at a concentration of 20 µg/ml. The IC50 value of DPPH
230
radical-scavenging activity was 6.27 ± 0.25 μg/ml (Table 1). Furthermore, DPPH
231
radical-scavenging activity was linearly correlated (positive) with the concentrations of
232
anthocyanin extracts from 0 to10 μg /ml, while ascorbic acid exhibited the activity from 2 to
233
30 μg/ml, respectively.
234
ABTS•+ is often used for in vitro determination of free radical activity and the relative
235
ability to scavenge the ABTS•+ radical has been compared with the standard trolox. Fig. 2B
236
shows a steady increase in ABTS radical-scavenging capacity, up to a concentration of 0.20
237
mg/ml, that is equivalent to 1.32 mg/ml of trolox. The IC50 value of ABTS radical-scavenging
238
activity was 90.3 ± 1.52 μg/ml.
239
It is noteworthy that both DPPH and ABTS radical-scavenging activities of the tested
240
extract were higher than those of VC (IC50 = 6.27 ± 0.25 μg/ml, 206 ± 2.37 μg/ml) which is
241
always considered as an excellent tool for determining the antioxidant activity of
242
hydrogen-donating antioxidants and of chain breaking antioxidants.
243 244
3.2.2. Reducing power activity
245
Reducing power is often used as an indicator of electron-donating activity, which is an
246
important mechanism for testing antioxidative action of phenolics. The reducing powers of
247
ART and ascorbic acid control are shown in Table 1 and Fig. 2C, respectively. As shown in
248
Fig.2C, the tested ART extract exhibited high reducing power in a dose-dependent manner,
249
but showed a slightly weaker ability compared with those of ascorbic acid. The value, raising
250
the absorbance at 700 nm to 0.5, of reducing power was 51.7 ± 0.74 μg/ml (Table 1).
251
Furthermore, the reducing power was linearly correlated (positive) with the concentrations of
252
anthocyanin extracts and ascorbic acid from 0 to 0.08 mg/ml. The corresponding correlation 11
253
coefficients were 0.996 for anthocyanin extracts (Y = 7.591X + 0.107) and 0.995 for ascorbic
254
acid (Y = 15.17 X + 0.026), respectively.
255 256
3.2.3. ORAC capacity test
257
The ORAC assay is the only antioxidant test that combines both inhibition time and degree
258
of inhibition into a single quantity. The assay uses a biologically relevant radical source, and is
259
also an assay where an added antioxidant competes with a substrate (fluorescein) for the
260
radicals generated by thermal decomposition of azo compounds, like AAPH, and inhibits or
261
retards substrate oxidation (Walton, Lentle, Reynolds, Kruger, & Mcghie, 2006). As
262
mentioned in 2.9, final ORAC values were expressed as μmol of trolox equivalents (TE) /mg
263
of ART, and a higher ORAC value indicated stronger antioxidant activity. As shown in Table
264
1, The ORAC value of the tested ART extract was 9.29 ± 0.08 μmol TE/mg, which was
265
significantly higher than that of ascorbic acid (1.79 ± 0.03 μmolTE/mg). The result indicated
266
that ART exhibited very good antioxidant activity.
267 268
3.3. HPLC analysis
269
Under the optimital HPLC separation condition, a satisfactory separation of the purified
270
anthocyanin extract of R. tomentosa was obtained and the HPLC chromatogram is shown in
271
Figure 3A.
272
The Major anthocyanins represented about 99% of the total peak area with regard to the
273
UV–Vis spectrum taken on-line during HPLC and chromatographic features. However, other
274
minor peaks were also detected which had percentage areas of less than 1%. No UV
275
absorbance maxima in the 310-320 nm range were detected, indicating no acylation of
276
anthocyanins with aromatic acids (Giusti & Jing, 2001).
277 12
278
3.4. Identification of compounds
279
The detailed MS data, including retention times, molecular ion peaks, MS2 fragments and
280
percent area at 520 nm, of all anthocyanins are summarized in Table 2. Fig.4 shows the
281
electrospray mass spectrum and the structures of the isolated anthocyanins.
282
Two major peaks, peak 2 and peak 5 (P2, P5) were obtained as amorphous red powder. As
283
shown in Table 2, P2 and P5 showed peaks at 449 m/z and 463 m/z from ESI–MS, which
284
were in accordance with the mass calculated for C21H21O11 (449.1) and C22H23O11 (463), on
285
the basis of the MS2 detected mass fragments at m/z 287 and m/z 301 related to the loss of one
286
hexose ([M-162]+) molecule, respectively, and the UV–Vis spectrum of the two compounds
287
showed the visible λmax to be 516 nm with the ratio of A440nm/Aλmax exceeding 0.20,
288
corresponding to cyanidin-3-O-glucoside and peonidin-3-O-glucoside (Cerezo et al., 2010;
289
Zhang, Xue, Yang, Ji, & Jiang, 2004). The NMR data of P2 and P5 were as follows:
290
Peak 2 (P2): 1H NMR (CF3COOD-CD3OD): δ6.64 (1H, d, J=1.2 Hz, H-6), δ6.86 (1H, d,
291
J=1.2 Hz, H-8), 8.22 (1H, dd, J = 8.8, 2.2 Hz, H-6´ ), 8.02 (1H, d, J = 2.2 Hz, H-2´ ), 7.04 (1H,
292
d, J = 8.8 Hz, H-5´ ), 8.98 (1H, s, H-4); 5.31 (1H, d, J = 7.8 Hz, H-1 glc), 3.69 (1H, m, H-2
293
glc), 3.56 (2H, m, H-3,5 glc), 3.46 (1H, m, H-4 glc), 3.73 (1H, dd, J = 12.2, 5.6 Hz, H-6b glc),
294
3.96 (1H, dd, J = 12.2, 2.2 Hz, H-6a glc). 13C NMR (CF3COOD-CD3OD): δ164.5 (C-2), 145.8
295
(C-3), 137.2 (C-4), 159.6 (C-5), 103.7 (C-6), 170.8 (C-7), 95.4 (C-8), 157.9 (C-9), 113.6
296
(C-10), 121.5 (C-1´ ), 118.7(C-2´ ), 147.6(C-3´ ), 156.0 (C-4´ ), 117.7 (C-5´ ), 128.4 (C-6´ ),
297
104.1 (C-1 glc), 75.0 (C-2 glc), 78.3 (C-3glc), 71.3 (C-4 glc), 79.0 (C-5 glc), 62.6 (C-6 glc).
298
Peak 5 (P5): 1H NMR ( CF3COOD-CD3OD): δ6.64 (1H, d, J=1.2Hz,,H-6), 6.88 (1H, d,
299
J=1.2 Hz,, H-8), 7.02 (1H, d, J = 8.4 Hz, H-5´ ), 8.21 (1H, dd, J = 8.4, 2.0 Hz, H-6´ ) , 8.16
300
(1H, d, J = 2.0 Hz, H-2´ ), 8.99 (1H, s, H-4), 3.99 (3H, s, OCH3); 5.31 (1H, d, J =8.0 Hz, H-1
301
glc), 3.65 (1H, m, H-2 glc), 3.56 (2H, m, H-3,5 glc), 3.46 (1H, m, H-4 glc), 3.73 (1H, dd, J =
302
12.0, 4.0 Hz, H-6b glc), 3.94 (1H, dd, J = 12.0, 2.0 Hz, H-6a glc). 13
13
C
303
NMR(CF3COOD-CD3OD): δ163.2 (C-2), 144.7 (C-3), 136.5 (C-4), 158.5 (C-5), 103.1 (C-6),
304
170.6 (C-7), 94.5 (C-8), 157.0 (C-9), 112.8 (C-10), 120.3 (C-1´), 114.5 (C-2´), 148.7 (C-3´),
305
155.7 (C-4´), 116.8 (C-5´), 128.1 (C-6´), 103.5 (C-1 glc), 75.1 (C-2 glc), 78.5 (C-3glc), 71.1
306
(C-4 glc), 78.8 (C-5 glc), 62.5 (C-6 glc), 57.0(OCH3).
307
The 1H NMR spectra of P2 showed the presence of two meta-coupled doublet (J = 1.2 Hz)
308
protons on the A-ring at δ 6.64 and 6.86 ppm, which were assigned to H-6 and H-8,
309
respectively. Two sets of doublet and one set of double doublet of an ABM system at δ 7.04
310
(1H, d, J = 8.8 Hz), 8.22 (1H, dd, J = 8.8, 2.2 Hz), 8.02 ppm (1H, d, J = 2.2 Hz) were observed
311
which were characteristic of H-5´, H-6´ and H-2´, respectively. The ring C was a flavanone
312
moiety. The proton signal at δ 3.46-5.31 (H-1-glc~H-6-glc) showed a D-glucopyranose moiety
313
which was assigned as β - configuration based on the large proton-coupling constants of its
314
anomeric proton δ 5.31 ppm (1H, d, J = 7.8 Hz, H-1´) (Byamukama, Kiremire, Andersen, &
315
Steigen, 2005). The cross peak at δ5.31/145.8 (H-1glc/C-3) in the HMBC spectrum of P2
316
indicated that the sugar moiety was attached to C-3, which was confirmed by Zarena and
317
Sankar (2012). Based on the above results and literature (Lee & ChoungL, 2011), P2 was
318
identified as cyanidin-3-O-glucopyranoside.
319
Comparing the 1H and 13C NMR spectra of P5 with those of P2, its spectral features were
320
closely similar to those of P2, except for the excess of the three-proton signal at δ 3.99 (3H, s)
321
in the 1H NMR spectra and one carbon signal at δ 57.0 in the
322
due to a methyl group attached to oxygen. Further support for this structure was obtained by
323
the mass spectrum of P5 displaying a [M]+ ion peak at m/z 463, corresponding to the excess of
324
an methylene moiety (14 mass units) from P2 ([M]+). Based on the above evidence, the
325
structure of P5, which differed from P2 by a methyl group attached to the H-3´ position
326
instead of a hydroxyl group attached to the H-3´ position on the B ring entity, was deduced to
327
be peonidin-3-O-glucoside and the chemical structure of P2 was further confirmed by 14
13
C NMR spectra, which was
328
comparison of the NMR and MS data with the literature (Fossen, Slimestad, Øvstedal, &
329
Andersen, 2002).
330
Peak 1 (P1): amorphous red powder; ESI-MS m/z 465; 1H NMR (CF3COOD-CD3OD):
331
δ6.66 (1H, d, J=1.2 Hz, H-6), 6.87 (1H, d, J=1.2 Hz, H-8), 7.74 (2H, d, J = 2.2 Hz, H-2´, 6´ ),
332
8.93 (1H, s, H-4); 5.34 (1H, d, J = 7.8 Hz, H-1 glc), 3.73 (1H, m, H-2 glc), 3.60 (2H, m, H-3,5
333
glc), 3.51 (1H, m, H-4 glc), 3.79 (1H, dd, J = 12.2, 5.4 Hz, H-6b glc), 3.94 (1H, dd, J = 12.2,
334
2.0 Hz, H-6a glc). 13C NMR(CF3COOD-CD3OD): δ164.2 (C-2), 144.6 (C-3), 136.8 (C-4),
335
159.4 (C-5), 103.1 (C-6), 170.8 (C-7), 95.4 (C-8), 157.8 (C-9), 113.0 (C-10), 121.5 (C-1´ ),
336
113.7(C-2´ ), 147.6(C-3´ ), 146.0 (C-4´ ), 147.9 (C-5´ ), 113.4 (C-6´ ), 103.5 (C-1 glc), 74.8
337
(C-2 glc), 78.0 (C-3 glc), 71.2 (C-4 glc), 78.8 (C-5 glc), 62.3 (C-6 glc).
338
The MS analysis of P1 (tR =11.75 min) showed an [M]+ ion at m/z 465 and a major
339
fragmentation in MS2 at m/z 303 (-162 amu) which would correspond to the monoglucoside of
340
delphinidin (Cerezo et al., 2010), The UV–Vis spectrum of this compound showed the visible
341
λmax to be 523 nm; the ratio of absorbance at 440nm to the absorbance at visible maximum
342
wavelength (A440nm/Aλmax ratio) for peak 1 was found to be 0.28, indicating that the compound
343
is delphinidin-3-O-glucoside (Longo & Vasapollo, 2006).
344
The 1H NMR spectra of P1 showed the presence of two meta-coupled doublet (J = 1.2 Hz)
345
protons on the A-ring at δ 6.66 and 6.87 ppm, which were assigned to H-6 and H-8,
346
respectively. One set of doublets of an AM system at δ 7.74 (2H, d, J = 2.2 Hz) was observed
347
which was characteristic of H-2´ and 6´.The analysis of the NMR spectral data also revealed
348
only a single glucose moiety with a proton signal at δ5.34 (1H, d, J = 7.8 Hz) coupled with the
349
C-3 aglycone at 144.6 ppm, indicating a D-glucopyranose moiety with a β-configuration
350
attached to the C-3 position. Thus the structure of P1 was elucidated to be
351
delphinidin-3-O-glucoside and was in agreement with the report of Pazmiño-Durán, Giusti,
352
Wrolstad and Glória (2001). 15
353
Peak 3 (P3): amorphous purple powder; ESI-MS m/z 479; 1H NMR (CF3COOD-CD3OD):
354
δ6.64 (1H, d, J=1.2 Hz, H-6), 6.89 (1H, d, J=1.2 Hz, H-8), 7.74 (1H, d, J = 2.2 Hz, H-6´ ),
355
7.90 (1H, d, J = 2.2 Hz, H-2´ ), 8.97 (1H, s, H-4), 3.99 (3H, s, OCH3); 5.33 (1H, d, J = 8.0 Hz,
356
H-1 glc), 3.69 (1H, dd, m, H-2 glc), 3.57 (2H, m, H-3,5 glc), 3.42 (1H, m, H-4 glc), 3.74 (1H,
357
dd, J = 12.2, 5.6 Hz, H-6b glc), 3.92 (1H, dd, J = 12.2, 2.0 Hz, H-6a glc).
358
NMR(CF3COOD-CD3OD): δ162.9 (C-2), 145.0 (C-3), 135.6 (C-4), 158.9 (C-5), 103.4 (C-6),
359
170.6 (C-7), 95.5 (C-8), 157.1 (C-9), 113.3 (C-10), 119.7 (C-1´ ), 109.6 (C-2´ ), 149.5 (C-3´ ),
360
146.0 (C-4´ ), 147.7 (C-5´ ), 113.0(C-6´ ), 103.5 (C-1 glc), 74.5 (C-2 glc), 78.0 (C-3glc), 71.0
361
(C-4 glc), 78.2 (C-5 glc), 62.6 (C-6 glc), 57.5( OCH3).
13
C
362
The ESI-MS spectrum of peak 3(tR = 13.73 min) was characterized by an ion signal at m/z
363
479 with an MS2 fragment at m/z 317 ([M-162]+) coinciding with the molecular formula
364
C22H23O12 of petunidin aglycone linked with a glucose moiety (Lin, Harnly, Pastor-Corrales,
365
& Luthria, 2008). The UV–Vis spectrum of pigment 3, taken on-line during HPLC, showed a
366
visible maximum at 526 nm.
367
Comparing the 1H and 13C NMR spectra of P3 with those of P1, its spectral features were
368
closely similar to those of P1, except for the excess of the three-proton signal at δ 3.99 (3H, s)
369
in 1H NMR spectra and one carbon signal at δ 57.5 in 13C NMR spectra due to a methyl group
370
attached to the H-3´ position instead of a hydroxyl group attached to the H-3´ position on the
371
B ring entity. By comparison with previous research data (Lee et al, 2009), P3 was assigned as
372
petunidin-3-O-glucoside.
373
Peak 4 (P4): amorphous red powder; ESI-MS m/z 433; 1H NMR (CF3COOD-CD3OD): δ
374
6.67 (1H, d, J =1.2 Hz, H-6), 6.94 (1H, d, J = 1.2 Hz, H-8), 7.05 (2H, d, J = 8.8 Hz, H-3´, 5´ ),
375
8.60 (2H, d, J = 8.8 Hz, H-2´, 6´ ), 9.08 (1H, s, H-4), 5.34(1H, d, J = 8.0 Hz, H-1 glc), 3.68
376
(1H, m, H-2 glc), 3.54 (2H, m, H-3,5 glc), 3.42 (1H, m, H-4 glc), 3.80 (1H, dd, J =12.2, 6.0
377
Hz, H-6b glc), 4.04 (1H, dd, J = 12.2, 2.0 Hz, H-6a glc). 13C NMR (CF3COOD-CD3OD): 16
378
δ164.8 (C-2), 145.8 (C-3), 137.4 (C-4), 159.9 (C-5), 103.7 (C-6), 170.9 (C-7), 95.5 (C-8),
379
158.1 (C-9), 114.5 (C-10), 121.5 (C-1´ ), 136.8 (C-2´ ), 118.6 (C-3´ ), 166.0 (C-4´ ), 118.7
380
(C-5´ ), 136.4 (C-6´ ), 104.1 (C-1 glc), 75.3 (C-2 glc), 78.5 (C-3glc), 71.3 (C-4 glc), 79.0 (C-5
381
glc), 62.6 (C-6 glc).
382
The molecular ion [M]+ at m/z 433 received from the ESI-MS analysis of P4 (tR =15.46 min)
383
confirmed the molecular formula, C21H21O10, for glucoside derivatives of pelargonidin
384
aglycone. The fragment ion [M+H-162]+ at m/z 271 was consistent with the structure of
385
pelargonidin with a loss of glucose moiety from pelargonidin-3-O-glucoside (Hong &
386
Wrolstad, 1990). The UV–Vis spectrum of this compound showed the visible λmax at 506 nm.
387
The 1H NMR and
13
CNMR spectra suggested that P4 contained a flavanone moiety; the
388
proton signals at δ 6.67(1H, d, J =1.2 Hz) and 6.94 (1H, d, J = 1.2 Hz) implied the presence of
389
two meta-coupled doublet protons on the A-ring which were assigned to H-6 and H-8,
390
respectively. Two sets of doublets of an AB system at δ 7.05 (2H, d, J = 8.8 Hz) and 8.60 (2H,
391
d, J = 8.8 Hz) were observed, which were characteristic of H-3´, H-5´ and H-2´, H-6´,
392
respectively. As P2, the large coupling constants (J = 8 Hz) for the anomeric protons (δ5.34,
393
1H, d, J = 8.0 Hz, H-1 glc) confirmed the presence of a β-D-glucosidic linkage in P4. By
394
comparison with the literature, P4 was further confirmed as pelargonidin-3-O-glucoside
395
(Pedersen, Andersen, Aksnes, & Nerdal, 1993).
396
Peak 6(P6): amorphous dark red powder; ESI-MS m/z 493; 1H NMR (CF3COOD-CD3OD):
397
δ6.74(1H, d, J = 1.9 Hz, H-6), 7.06 (1H, d, J = 1.9 Hz, H-8), 7.96 (2H, d, J = 2.2 Hz, H-2´, 6´ ),
398
8.96(1H, s, H-4), 3.90 (6H, s, 2×CH3); 5.37 (1H, d, J = 7.8 Hz, H-1 glc), 3.64(1H, m, H-2 glc),
399
3.74 (1H, m, H-3 glc), 3.79 (1H, m, H-5 glc), 3.43 (1H, m, H-4 glc), 3.51 (1H, dd, J = 12.4,
400
4.8 Hz, H-6b glc), 3.96 (1H, dd, J = 12.4, 2.2 Hz, H-6a glc). 13C NMR (CF3COOD-CD3OD):
401
δ163.0 (C-2), 145.0 (C-3), 136.2 (C-4), 158.9 (C-5), 103.1 (C-6), 170.5 (C-7), 95.1 (C-8),
402
157.4 (C-9),112.9 (C-10), 120.0 (C-1´ ), 110.5 (C-2´ ), 149.1 (C-3´ ), 146.7 (C-4´ ), 149.2 17
403
(C-5´ ), 111.0(C-6´ ), 103.8 (C-1 glc), 74.6 (C-2 glc), 78.0 (C-3glc), 70.9 (C-4 glc), 77.8 (C-5
404
glc), 67.3 (C-6 glc), 57.3(2×OCH3).
405
The last peak in the chromatogram was peak 6, with the MS and MS2 profiles from ESI-MS
406
spectra showing strong ion peaks at m/z 493 and 331 for peak 6 (tR = 15.91 min) coinciding
407
with the molecular ion of malvidin-3-O-glucoside with a loss of a glucose moiety (Li, Wang,
408
Guo, & Wang, 2011). Based on the spectrum data and comparing with previously reported
409
data (Alcalde-Eon, Escribano-Bailón, Santos-Buelga, & Rivas-Gonzalo, 2006), P6 was
410
identified as malvidin-3-O-glucoside.
411
As shown in Figure 3A, there are six principal anthocyanin peaks, and two major peaks
412
(peak 2 and peak 5) with concentrations of 47% (29.4 ± 0.39 mg/100g) and 34 % (21.3 ± 0.33
413
mg/100g) of the total peak area with retention times of 13.20 and 15.46 min, respectively,
414
(Table 2). The elution times of the other four peaks with the concentration of 3.35% (peak 1,
415
2.07 ± 0.03 mg/100g), 5.42% (peak 3, 3.51 ± 0.14 mg/100g), 0.85% (peak 4, 0.49 ± 0.05
416
mg/100g), 8.86% (peak 6, 5.69 ± 0.16 mg/100 g) were 11.75 (peak 1), 13.73 (peak 3), 14.78
417
(peak 4), 15.90 (peak 6) min. The final structures of the six compounds isolated and identified
418
are shown in Figure 3B.
419
Based on the available literature, there have been only a few reports in previous studies
420
regarding the extraction and preliminary qualitative research of the anthocyanins in R.
421
tomentosa fruits. This is the first time that all the major anthocyanins were systematically
422
isolated and identified from R.tomentosa fruits.
423 424
4. Conclusions
425
The total anthocyanin content of the purified Rhodomyrtus tomentosa (Ait.) Hassk fruits
426
extract was 62.8 ± 1.2 mg/100 g of freeze-dried weight of R.tomentosa fruits and it possessed
427
excellent in vitro free radical-scavenging activity. Pure anthocyanins were isolated and 18
428
analyzed by ESI-MS and NMR spectroscopy. The six anthocyanin structures characterised
429
were
430
pelargonidin-3-O-glucoside, peonidin-3-O-glucoside, and malvidin-3-O-glucoside. As a result,
431
this study has systematically documented, for the first time, the presence of six anthocyanin
432
derivatives from the tested extract. Fruits of R. tomentosa also contain important amounts of
433
other phenolics, amino acids and ascorbic acid. These phytochemicals may partially explain
434
the diverse bioactive properties of this plant. Therefore, R. tomentosa could be a source of
435
functional substances for human health and the food industry. Further investigation is to
436
compare the differences of the type and content of anthocyanins in Rhodomyrtus tomentosa of
437
different regions in China to provide a theoretical support for further development of this
438
resource.
delphinidin-3-O-glucoside,
cyanidin-3-O-glucoside,
petunidin-3-O-glucoside,
439 440
Acknowledgements
441
The authors are grateful to the National Natural Science Foundation of China (No.
442
31201416, 31000759 and 31101222) and Technology Program (Nos. 2011BAD23B01) for
443
their financial support.
444 445
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Figure captions:
529
Fig. 1 The fruit and flower of Rhodomyrtus tomentosa (Ait.) Hassk collected in Guangdong
530
province.
531
Fig. 2 Antioxidant activities of ART extracted from Rhodomyrtus tomentosa fruit determined
532
by DPPH and ABTS free radical-scavenging assays (A and B) and reducing power assay(C).
533
Fig. 3 HPLC chromatogram (A) showing the six anthocyanin profiles of Rhodomyrtus
534
tomentosa extract monitored at 520 nm. (Table 2 shows the peak identification) and the
535
structures of the anthocyanins (B) isolated from the fruits of Rhodomyrtus tomentosa.
536
Fig. 4 Electrospray mass spectrum of identified anthocyanins. Peak 1:
537
delphinidin-3-O-glucoside; Peak 2: cyanidin-3-O-glucoside; Peak 3: petunidin-3-O-glucoside;
538 539
Peak 4: pelargonidin-3-O-glucoside; Peak 5: peonidin-3-O-glucoside; Peak 6: malvidin-3-O-glucoside.
23
540
Table 1 Antioxidant activities of anthocyanins (ART) extracted from Rhodomyrtus tomentosa
541
and ascorbic acid using the DPPH• assay, ABTS+• assay, reducing power assay and ORAC test IC50 / DPPH•
IC50 / TEAC
A700nm=0.5/Reducing
ORAC value
(μg/ml)1
(μg/ml)2
Power(μg/ml)3
(μmolTE /mg)
ART
6.27 ± 0.25b
90.3 ± 1.52b
51.7 ± 0.74a
9.29 ± 0.08a
ascorbic acid
17.4 ± 0.31a
206 ± 2.37a
31.3 ± 0.93b
1.79 ± 0.03b
Samples
542
All the trials were performed in triplicate (n = 3) and all the data represent the means ±
543
standard deviation (n > 3). Data in the same column with different letters are significantly
544
different (p < 0.05).
545
1
546
needed to decrease the absorbance at 517nm by 50%.
547
2
548
decrease the absorbance at 734nm by 50%.
549
3
550
the absorbance at700nm to 0.5.
The antioxidant activity was calculated as the concentration of the test sample
The antioxidant activity was evaluated as the concentration of the test sample required to
The antioxidant activity was evaluated as the concentration of the test sample needed to raise
24
551
Table 2 LC-MS characteristics of anthocyanins separated from Rhodomyrtus tomentosa: retention time, wavelengths of maximum absorption
552
(λmax), molecular ion, fragmentation pattern and tentative identification
553
a, b
The fragment ions are shown in order of their relative abundance.
Peak.No.
Elution
Peak
λmax
Molecular
Mass loss
MS2 of
Peak
Contents
time(min)
Area(%)
(nm)
ion[M+]，m/za
(M+H+)-MS2
[M+]，m/zb
assignment
(mg/100g
(520nm))
m/z
dry fruits)
1
11.75
3.35
523/277
465
-162
302.9
Delphinidin-3-O-glucoside
2.07 ± 0.03
2
13.20
47.27
516/280
449/286.9
-162
286.9
Cyanidin-3-O-glucoside
29.4 ± 0.39
3
13.73
5.42
526/277
479/316.9
-162
316.9
Petunidin-3-O-glucoside
3.51 ± 0.14
4
14.78
0.85
516/281
433/271.0
-162
271.0,415.1
Pelargonidin-3-O-glucoside
0.49 ± 0.05
5
15.46
34.22
506/279
463/300.9
-162
300.9
Peonidin-3-O-glucoside
21.3 ± 0.33
6
15.90
8.86
527/277
493/331.0
-162
331.0
Malvidin-3-O-glucoside
5.69 ± 0.16
25
554
555
Fig. 1
26
DPPH•-Scavenging Capacity (%)
556
A 100 80 60 ART Vc
40 20 0 0
4
8
12
16
Concentration
557
mg trolox equivalent /mg
B
20
24
28
32
0.18
0.21
( µ g/ml )
1.4 1.2
ART Vc
1.0 0.8 0.6 0.4 0.2 0.0 0.00
0.03
0.06
0.09
0.12
Concentration(mg/ml)
27
0.15
Reducing Power A700 nm
C
558
2.0 ART Vc
1.6 1.2 0.8 0.4 0.0 0.00
0.02
0.04
0.06
Concentration(mg/ml)
559
Fig. 2
28
0.08
0.10
560
561
Fig. 3
29
562
563
Fig. 4
30
564
Research Highlights
565
1. All the major anthocyanins were isolated and identified from R.tomentosa fruits.
566
2. Cyanidin-3-O-glucoside was considered as the most abundant antocyanin.
567
3. The purified anthocyanin extract showed strong antioxidant activities.
568 569
31