Comparative study on the chemical structure and in vitro anti

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Bioactive Constituents, Metabolites, and Functions

Comparative study on the chemical structure and in vitro anti-proliferative activity of anthocyanins in purple root tubers and leaves of sweet potato (Ipomoea batatas) Vijayakumar R. Vishnu, Raveendran S Renjith, Archana Mukherjee, Shirly Raichal Anil, Janardanan Sreekumar, and Jyothi Narayanan Alummoottil J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b05473 • Publication Date (Web): 11 Feb 2019 Downloaded from http://pubs.acs.org on February 11, 2019

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Journal of Agricultural and Food Chemistry

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Comparative study on the chemical structure and in vitro anti-proliferative

2

activity of anthocyanins in purple root tubers and leaves of sweet potato

3

(Ipomoea batatas)

4 5

Vijayakumar R Vishnu1, Raveendran S Renjith1, Archana Mukherjee2, Shirly Raichal

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Anil2, Janardanan Sreekumar3, Alummoottil N Jyothi1*

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1Division

Sreekariyam, Thiruvananthapuram, Kerala, India

8 9

2Division

of Crop Improvement, ICAR-Central Tuber Crops Research Institute, Sreekariyam, Thiruvananthapuram, Kerala, India

10 11

of Crop Utilization, ICAR-Central Tuber Crops Research Institute,

3Section

of Extension and Social Sciences, ICAR-Central Tuber Crops Research Institute, Sreekariyam, Thiruvananthapuram, Kerala, India

12 13 14 15 16

*Address for Correspondence: Dr. A. N. Jyothi

17

Division of Crop Utilization, ICAR-Central Tuber Crops Research Institute,

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Sreekariyam, Thiruvananthapuram - 695017, Kerala, India.

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Ph: +91 471 2598551, Fax: +91 471 2590063

20

Email: [email protected]

21 22

Running title:

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Structure-activity relationship of sweet potato anthocyanins

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1 ACS Paragon Plus Environment


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ABSTRACT: The structure and in vitro anti-proliferative activity of anthocyanins in the root

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tubers of a sweet potato variety cv Bhu Krishna and the purple leaves of a promising

27

accession S-1467 were studied with the objectives of understanding the structure-activity

28

relationship and to compare the leaf and tuber anthocyanins. The chemical structure of

29

anthocyanins was determined by HR-ESI-MS analysis. FRET-based caspase sensor probe

30

had been used to study the anti-proliferative property and analysis of cell cycle was done

31

after staining with propidium iodide and subsequent fluorescence-activated cell sorting.

32

Structurally the anthocyanins in root tubers were identical to those in leaves; but there was a

33

difference in the proportion of various aglycones present in both. This has lead to

34

distinguishable differences in the anti-proliferative activity of leaf and tuber anthocyanins to

35

various cancer cells. All the nine anthocyanins were found in acylated forms in both tuber

36

and leaves. However, peonidin derivatives were major anthocyanins in tubers (33.98±1.41

37

mg) as well as in leaves (27.68±1.07mg). The cyanidin derivatives were comparatively

38

higher in leaves (20.55±0.91mg) than in tubers (9.44±0.94 mg). The tuber and leaf

39

anthocyanins exhibited potential anti-proliferative properties to MCF-7, HCT-116 and HeLa

40

cancer cells and the structure of anthocyanins had a critical role in it. The leaf anthocyanins

41

exhibited significantly higher activity against colon and cervical cancer cells, whereas tuber

42

anthocyanins had a slightly greater effect against breast cancer cells.

43

KEYWORDS: Anthocyanins, sweet potato, HR-ESI-MS, anti-proliferative activity, cell

44

cycle

45 46 47 48 49


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INTRODUCTION

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Root and tubers serve as secondary staple for approximately one-fourth of the world’s

52

population in the tropics. These crops play a substantial role in food security and nutrition

53

apart from their climate resilience. Most of the tuber crops are potential sources of bioactive

54

phytochemicals including flavonoids.1,2 Purple varieties of sweet potato contain anthocyanins

55

with high antioxidant property. Previous studies in mice have shown the preventive effect of

56

these anthocyanins on liver damage due to alcoholism.3 It has a promising clinical efficacy

57

against hyperuricemia also.4 These anthocyanins also have prebiotic-like activity and

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generate fatty acids necessary for intestinal and colon health.5

59

More than 15 acylated anthocyanins including Caffeoylquinic acid with potential biological

60

activity, were identified in the root tubers of sweet potato depending on the cultivars.6,7

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Ayamurasaki, a purple tuber flesh colored sweet potato variety from Japan is one of the well-

62

known anthocyanin sources and the major anthocyanins exist in acylated form with ferulic

63

acid, caffeic acid and p-hydroxybenzoic acid.8 The low-density lipoproteins (LDL), which are

64

considered as “bad” cholesterol, are reported to be protected against oxidation by

65

anthocyanins from Ayamurasaki more effectively than those from other sources.9 These

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anthocyanins can also restrain the progression of atherosclerosis and increase in oxidative

67

stress.9Anthocyanins are reported to have strong antioxidant activity, prevent the growth of

68

tumor cells and cause apoptosis in these cells.10,11 Cyanidin has been reported as the major

69

aglycone/anthocyanidin in the leaves of three sweet potato varieties in Japan.12

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The cultivar, Bhu Krishna released from ICAR-CTCRI, Thiruvananthapuram, Kerala, India

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has a rich content of anthocyanins in its purple-fleshed root tubers.13The antibacterial activity

72

of the extracts of these root tubers has been reported.14The sweet potato accession, S-1467 in

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the germplasm is peculiar in having all its leaves purple-colored while possessing white-

74

fleshed root tubers. A preliminary study showed that these leaves are good sources of



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anthocyanins. At present, no literature is available on systematic investigations on the

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structure and activity of purple leaf anthocyanins isolated from sweet potato. Also, studies

77

concerning the comparison of structure and activity of anthocyanins in sweet potato leaves

78

and tubers are also scanty. Therefore, the current study had a focus on finding out and

79

comparing the structure of anthocyanins isolated from the purple-fleshed root tubers of sweet

80

potato cultivar Bhu Krishna and the leaves of Acc. S-1467 and also to evaluate the in vitro

81

anti-proliferative activity of these anthocyanins in relation to structure.

82

MATERIALS AND METHODS

83

Chemicals

84

Methanol (99.5%), trifluoroacetic acid (99.5%) and ethyl acetate (99.5%) were obtained from

85

Merck India Pvt Ltd. (Mumbai, India). The resins, Sephadex LH-20 and Amberlite XAD-7

86

were procured from Sigma Aldrich (St. Louis, USA). Acetonitrile (CH3CN, 99.8%) and

87

water (Vetec, India), cyanidin-3-O-glucoside (99.9%) and peonidin-3-O-glucoside (99.9%)

88

(Sigma Aldrich, St. Louis, USA) were employed for HPLC studies. Other chemicals used

89

were of analytical reagent (AR) grade.

90

Selection of extraction solvent

91

Fresh root tubers of the sweet potato variety, Bhu Krishna and leaves of the accession, S-

92

1467 were collected from the experimental farm of ICAR-Central Tuber Crops Research

93

Institute, Kerala, India. The tuber and leaf samples (5 g each) were homogenized for 1 minute

94

by using a Polytron homogenizer (PT-MR 2100, Switzerland) in different extraction solvents

95

of methanol-trifluoroacetic acid (TFA) (99.5: 0.5), ethanol-TFA (99.5:0.5), Methanol-TFA-

96

water (80:19.5:0.5), and ethanol-TFA-water (80:19.5:0.5). The anthocyanins rich supernatant

97

was separated from residue by filtration and extraction was continued till the residue became

98

colorless. The filtrate was treated with 6M HCl solution and HPLC studies have been done

99

with this acid treated extract. Different homogenization time (1 to 3 minutes) and


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100

sample/solvent ratio (1:2, 1:4 and 1:6, v/v) were used to study the extraction efficiency.15

101

The sample was homogenized for with the selected solvent. Thus, both the tuber and leaves

102

(5 g each) was homogenized with 10, 20 and 30 ml of methanol acidified with 0.5% TFA.

103

Further hydrolysis and HPLC were carried out using this homogenate.

104

Purification of anthocyanins

105

Fresh samples of root tubers and leaves were extracted and extraction solvent was removed

106

with the aid of a rotary flash evaporator (Buchi-multivapour, BUCHI Labortechnik AG,

107

Switzerland) at 30C and the concentrate was again dissolved in a little amount of acidified

108

water. The aqueous extract was then subjected to partition with ethyl acetate for removing the

109

less polar impurities including chlorophyll in the case of leaves. Purified extract was then

110

subjected to column chromatography using Amberlite XAD-7 HP with a mesh size of 20-60

111

(Sigma Aldrich, USA) and the mobile phase was double distilled water. The elution of

112

adsorbed anthocyanins was done with methanol containing 0.5% TFA. The purified extract

113

was concentrated, lyophilized and crystallized at trap temperature of -90C for 3 h.

114

HPLC analysis of purified anthocyanins

115

Analytical reversed-phase ultra-high performance liquid chromatography (UFLC) was

116

performed in accordance with a reported procedure with slight modifications.16 The

117

instrument, Shimadzu UFLC was connected with an LC-20 AD pump, column oven (CTO

118

10AVP) and SPD-10A VP UV-Vis detector (Shimadzu, Japan). A reverse phase Varian

119

Pursuit XRS 5 C18 column (250×4.6 mm) and a guard column (Meta guards, 4.6mm pursuit

120

XRs 5u C18) were used for the analysis. The solvents used were water (A) and acetonitrile

121

(B), both containing 0.5% TFA. Initially, 90% A and 10% B was used for elution which was

122

followed by a 10 min linear gradient elution with 14% B. Subsequently, isocratic elution (10-

123

14 min) and linear gradient elution were performed under the following conditions. The

124

concentration of B increased during the process up to 16% for 14 to 18 min, 18% for 18 to 5 ACS Paragon Plus Environment


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22 min, 23% for 22 to 26 min, 28% for 26-31 min, and 40% for 31- 32 min. Then it was

126

reduced to 10% for 32-35 min and finally it was maintained for 35-38 min. Twenty μL of

127

sample solution was injected into the column and the flow rate was maintained at 1.0

128

mL/min. The analysis was done at a wavelength of 520nm.

129

Acid hydrolysis of anthocyanins

130

Acid hydrolysis of anthocyanins was carried out according to a previously reported procedure

131

with slight modification.16 The aqueous solution of anthocyanins, after

132

extraction, was made up to 10 ml with deionized water and then 6 M HCl (4 ml ) was added

133

to it. Then hydrolysis of anthocyanins was carried out under nitrogen atmosphere for 45 min

134

at 90°C. The hydrolysate was cooled in an ice-bath; the pH was adjusted to 3.0 by using 20%

135

solution of KOH and then concentrated under reduced pressure. Two milliliters of 0.01% HCl

136

was added to the residue and filtered using a 0.45 μm membrane filter. The hydrolyzed

137

anthocyanins were subjected to HPLC and LC-MS analyses.

138

Quantitative determination

139

The fresh root tubers and leaves were finely chopped and 10g each was weighed out into a

140

screw-cap bottle. It was then extracted with 10ml of the mixture of acidified methanol and

141

kept in refrigerator at 4C for 24 h. The extract was then filtered and the process is repeated

142

till the residue was colorless. The extracts were combined; volume was noted and stored in a

143

refrigerator after sealing. Five replicate samples were made in each case. Before injection

144

into the HPLC system, the extracts were filtered by using a 0.45 μm Millipore membrane

145

filter. The stock solution was diluted with 0.5% TFA in methanol, yielding concentrations of

146

5, 10, 20, 40, 80 and 160 µg/mL as calibration standards. Peonidin-3-glycoside and cyanidin-

147

3-glucoside were taken as standard reference materials to determine the quantitative amounts

148

of peonidin and cyanidin derivatives in the samples and were presented as milligram

149

equivalents per 100 g fresh weight of the sample.

ethyl acetate


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Fractionation of anthocyanins and HR-ESI-MS analysis

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Five hundred milligrams of purified anthocyanins were fractionated by gel filtration

152

chromatography using Sephadex LH 20 and gradient elution was performed using acidified

153

water and methanol mixture by low-pressure liquid chromatography (BIO-RAD, BioLogic

154

LP). Initially, 5% methanol was used for elution at a flow rate of 0.5 ml/min. For complete

155

elution, four different concentrations of methanol, i.e., 5, 10, 15 and 20% were used.

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Anthocyanins appeared as different distinguishable bands during the elution. The major

157

anthocyanin was eluted using 10% methanol. Fractionated anthocyanins were subjected to

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HR-ESI-MS analysis using a Thermo scientific instrument coupled with an orbit trap mass

159

analyzer equipped with an auto sampler and HPLC. The column used was C18 (hypersil gold

160

50×2.1mm). The composition of mobile phase was 97% of methanol and 3% of 0.1% formic

161

acid in de-ionized water. The flow rate was maintained at 150µl per minute. The sample

162

injection volume was 2µl and other conditions were: scan range, m/z 100 to 1500, nitrogen

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drying gas pressure 40psi, capillary voltage of 30V, temperature 300°C, and analysis time 5

164

minutes.

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Animal cell culture

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Cell lines

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The human mammarian epithelial cells (MCF-10A, Sigma-Aldrich) were stored in MEBM

168

with growth factors and kept at 37°C in a CO2 incubator with 5% CO2 atmosphere. The

169

medium was replaced every four days. The human cancer cell lines used for the study were

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MCF-7 (breast cancer cells), cervical cancer cells, HeLa and HCT-116 (colon cancer cell

171

lines), and were procured from American Tissue Culture Collection (ATCC) and maintained

172

as per standard protocol. The cells were maintained at 37°C in RPMI1640/DMEM which

173

contains 10% FBS and kept at 5% CO2 in an incubator. The medium was replaced in every

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four days.



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Apoptotic studies

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Live cell staining with Hoechst 33342

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Hoechst staining was done in human mammary epithelial cells (MCF-10A). For this,

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apoptosis was induced in the cells which were grown in 96 well plates. About 60 ml of the

179

medium was removed from the wells and the same amount of 0.5µg/ml of the diluted dye

180

was added to it. The cells were kept at 37ºC in a 5% CO2 incubator for 10 min. About 60 ml

181

of the medium was removed from the wells and observed under the fluorescent microscope.

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After washing with fresh medium, the wells were added with anthocyanins at 100, 200 and

183

400 μg/ml concentrations and maintained at 37ºC and 5% CO2 until imaging. For Hoechst

184

imaging, Epi-Fluorescent Microscope TiE (Nikon, Japan) was used and the cells were

185

observed using DAPI filter sets. Retiga Exi Camera (Q imaging) with NIS element software

186

was used for capturing images.

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Study of apoptosis using FRET-based caspase sensor probe

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The effect of treatment with purple leaf and tuber anthocyanins on inducing apoptosis in

189

various cancer cells was studied. The cells indicating FRET-based caspase sensor (ECFP-

190

DEVD-EYFP) were seeded in 96 well plates and incubated at 37ºC in a 5% CO2 incubator

191

for 24 hours. Upon reaching 50-70% confluency, the cells were treated with purple leaf and

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tuber anthocyanins (200 and 400 μg/ml) containing imaging medium and maintained at 37ºC,

193

in CO2 atmosphere until imaging. The cell images were obtained by using BD pathway™

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435 Bio imager (BD Biosciences, USA) using the Attovision™ software. For imaging, the

195

single excitation wavelength of 438±12 nm was used, whereas emission was collected in two

196

wavelengths, 483±15 nm and 542±27 nm respectively, for ECFP and EYFP. Images were

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captured using a 20×dry objective with the numerical aperture of 0.75. Cells showing FRET

198

loss were considered as apoptotic cells. The cells with caspase activation as identified from


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ratio image were counted from four separate image fields to calculate the percentage of cells

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with caspase activation.

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Flow cytometry

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The growth inhibition mechanism of test cell lines by anthocyanins was studied by analyzing

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the cell cycle distribution using flow cytometry.18 The HeLa, HCT-116 and MCF-7 cells

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(1×106) were plated and left for 24h to facilitate attachment, which was followed by

205

treatment with anthocyanins (100 and 200 μg/ml) for 48h. A solution of phosphate buffered

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saline was used to wash the cells after treatment and then kept overnight with 70% ethanol at

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20°C. This was followed by incubation at 37°C in dark for 30 min with propidium iodide (PI)

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(PBS containing 10 µl of 1 mg/ml PI, 0.03% of NP-40 and 5µl of 10 mg/ml RNase A). The

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cells were then analyzed by using a flow cytometer (BD FACS Aria II, BD Biosciences, San

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Jose, USA).

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Statistical analysis

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The average values of three replications were taken and reported. Single factor analysis of

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variance (ANOVA) of data was done by using the package SAS 9.3. TUKEY's Honest

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Significant Difference test was done to understand post-hoc correlations of mean values.

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RESULTS AND DISCUSSION

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Comparison of extraction solvents

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The extraction efficiency of anthocyanins was compared with various solvents. Both purple-

218

colored tuber and leaves contained high concentration of cyanidin and peonidin in methanol-

219

TFA extract (table 1).

220 221

Table 1. Extraction efficiency of anthocyanins in different solvent systems

Sample

Extraction solvent

Solvent ratio

Amount of

Amount of

cyanidin

peonidin

derivatives /100

derivatives /100g 9

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g fresh weight

fresh weight

Methanol - TFA

99.5:0.5

9.44 ± 0.94l

33.98 ± 1.41a

Ethanol - TFA

99.5:0.5

8.21 ± 0.82m

31.63 ± 1.12b

Methanol - Water - TFA

80:19.5:0.5

7.59 ± 0.55n

30.19 ± 0.97c

Ethanol - Water - TFA

80:19.5:0.5

7.12 ± 0.69o

30.03 ± 1.17d

Methanol - TFA

99.5:0.5

20.55 ± 0.91i

27.68 ± 1.07e

Ethanol - TFA

99.5:0.5

19.61 ± 1.12j

25.14 ± 1.31f

Methanol - Water - TFA

80:19.5:0.5

19.17 ± 0.67k

24.98 ± 0.95g

Ethanol - Water - TFA

80:19.5:0.5

19.11 ± 0.77l

23.18 ± 1.05h

Tuber

Leaf

222 223 224

*Mean

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On the basis of these results, it was found that acidified methanol with 0.5% TFA was more

226

efficient in the extraction of anthocyanins and hence it was used for further analysis of

227

anthocyanins. The anthocyanins content was significantly higher at a sample/solvent ratio of

228

1:6. For the extraction of anthocyanins, the samples were homogenized with acidified

229

methanol for 1, 2 and 3 minutes. Since there was no significant change in the quantity of

230

anthocyanins extracted, the minimum time of 1 minute was used for homogenization in

231

further study.

232

HPLC analysis and quantitative comparison of anthocyanins

233

The HPLC chromatogram at 520 nm showed that both the purple leaves of S-1467 and the

234

root tubers of Bhu Krishna consisted of nine acylated anthocyanins each (figure 1a and 1b),

235

which were peonidin and cyanidin derivatives. All the anthocyanins were structurally similar

236

in both cases but the only difference was in their proportion. In both cases, the anthocyanin

237

corresponding to peak 8 was found to be the major contributing anthocyanin. The HPLC

238

profile of the acid hydrolyzed leaf and tuber anthocyanins indicated that all the nine

239

anthocyanins produced only two different aglycones (figure 1c and 1d). The HPLC

240

chromatograms of these aglycones were similar to those of acid-hydrolyzed cyanidin-3-O-

values with at least one common letter in the superscript are not statistically significant using TUKEY's Honest Significant Difference


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glucoside as well as peonidin-3-O-glucoside standards (figure 1e and 1f, respectively). Even

242

though the constituent anthocyanins vary with variety as well as the plant part which contains

243

anthocyanins even within the same crop, it was interesting to note that the two different sweet

244

potato accessions possessed the same number and type of anthocyanins-root tubers in one

245

case and leaves in the other.

246

The total anthocyanin content in Bhu Krishna root tubers as quantified by HPLC was 43.4

247

mg/100g fresh wt. of peonidin-3-O-glucoside equivalent (table 2). This yield was slightly less

248

than that of the variety 'Ayamurasaki', which is one of the highest anthocyanin containing

249

sweet potato variety (59 mg of peonidin-3-caffeoylsophoroside-5-glucoside equivalents/100g

250

fresh wt.).19,20 In the leaves of S-1467, the anthocyanin content was higher than that in root

251

tubers of Bhu Krishna and it was 48.2 mg/100g fresh wt. of peonidin-3-O-glucoside. The

252

total monomeric anthocyanins in four American breeding clones of sweet potato was in the

253

range of 24.6-45.1 mg cyanidin-3-glucoside/100g fresh weight.21 A sweet potato breeding

254

variety called 'Stokes Purple' popular in North Carolina was reported to contain about 57.5

255

mg anthocyanins/100 g fresh wt.22

256

Individual anthocyanins quantified by HPLC showed that peonidin derivatives were 79.2%

257

and 57.3% and cyanidin derivatives were 21.8% and 42.7%, respectively, in tubers and leaves

258

(table 2). The total peonidin/cyanidin (Peo/Cy) ratio in leaf anthocyanins was 1.34, whereas

259

in the tuber, it was 3.63. Earlier studies have shown a wide variation in Peo/Cy ratio in sweet

260

potato tuber anthocyanins. The Peo/Cy ratio of some of the Japanese purple sweet potato

261

varieties viz., Chiranmurasaki, Tanegashimamurasaki, Nakamurasaki, and Purple Sweet were

262

reported as 4.05, 0.04, 0.10 and 4.52, respectively.23

263 264 265



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Table 2. High-resolution MS data and anthocyanin quantity in purple root tubers of sweet potato cv Bhu Krishna and leaves of Acc. S-1467

266 267

Peak no.

Compound

[M+]

Fragment ions

Quantity

(m/z)

(m/z)

(mg/100g FW) Leaves

1a

Cy-3-O-(6-p-

Tuber

893

731, 449, 287

0.58 ± 0.12o

0.32 ± 0.05p

907

745, 463, 301

0.81 ± 0.14n

0.76± 0.10no

1097

935, 449, 287

4.10 ± 0.18g

2.16 ± 0.05k

1055

893, 449, 287

15.0 ± 0.39b

3.71 ± 0.16h

1111

949, 449, 287

0.87 ± 0.09n

3.25 ± 0.11i

1111

949, 463, 301

1.51 ± 0.11l

8.79 ± 0.19d

949

787, 463, 301

4.99 ± 0.29f

2.70 ± 0.08j

1069

907, 463, 301

19.3 ± 0.40a

14.0 ± 0.35c

1125

963, 463, 301

1.07 ± 0.12m

7.73 ± 0.10e

hydroxybenzoylsoph)-5-O-glc 2b

Peo-3-O-(6-phydroxybenzoylsoph)-5-O-glc

3a

Cy-3-O-(6,6‴-dicaffeoylsoph)-5O-glc

4a

Cy-3-O-(6-caffeoyl-6‴-phydroxybenzoylsoph)-5-O-glc

5a

Cy-3-O-(6-caffeoyl-6‴feruolylsoph)-5-O-glc

6b

Peo-3-O-(6, 6‴-dicaffeoylsoph)5-O-glc

7b

Peo-3-O-(6-caffeoylsoph)-5-Oglc

8b

Peo-3-O-(6-caffeoyl-6‴- phydroxybenzoylsoph)-5-O-glc

9b

Peo-3-(6-caffeoyl-6‴feruolylsoph)-5-glc

268 269 270 271

*Mean

values with at least one letter common are not statistically significant using TUKEY's Honest Significant Difference. amg cyanidin 3-glucoside equivalents/100g FW; bmg peonidin 3-glucoside equivalents/100g FW. 12 ACS Paragon Plus Environment

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HR-ESI-MS Spectroscopy for structural analysis of anthocyanins

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High resolution-ESI-mass spectra were obtained for anthocyanins isolated from the purple

274

tuber and leaves (Mass spectra are given as supplementary information). Figure 2 represents

275

the structure of different anthocyanins. With positive ionization, an anthocyanin (Compound

276

1, tR17.3), cyanidin-3-O-(6-p-hydroxybenzoylsophoroside)-5-O-glucoside (Cy-3-O-(6-p-

277

hydroxybenzoylsoph)-5-O-glc) produced the peak of [M]+ at m/z 893, and peaks

278

corresponding to the fragment ions [Cy-3-O-(6-p-hydroxybenzoylsoph)]+ , [Cy-3-O-glc]+

279

and [Cy]+ were at m/z 731, 449 and 287, respectively. Compound 2 (tR 22.9), Peo-3-O-(6-p-

280

hydroxybenzoylsoph)-5-O-glc had [M]+ ion peak at m/z 907 and three fragment ion peaks at

281

m/z 745,463 and m/z 301 (table 2). The base peak at m/z 301 showed the presence of

282

peonidin [Peo]+ as aglycone. Compound 3 (tR30.2), Cy-3-O-(6,6‴- dicaffeoylsoph)-5-O-glc

283

had [M] + ion peak at m/z 1097 and the first fragmentation peak at m/z 935 was that of [Cy-3-

284

O-(6,6‴- dicaffeoylsoph)]+. Compound 4, the second major anthocyanin in the purple leaves,

285

with tR 30.6 was Cy-3-O-(6-caffeoyl-6‴-p-hydroxybenzoylsoph)-5-O-glc, which showed

286

[M]+ ion peak at m/z 1055 and the first fragmentation peak at 893 representing [Cy-3-O-(6-

287

caffeoyl-6‴-p-hydroxybenzoylsoph)]+. Compound 5 with tR 31.8, Cy-3-O-(6-caffeoyl-6‴-

288

feruolylsoph)-5-O-glc produced [M]+ ion peak at m/z 1111 and its first fragmentation peak

289

was observed at m/z 949, [Cy-3-O-(6-caffeoyl-6‴-feruolylsoph)]+. The [M]+ ion peak of the

290

second major anthocyanin in the sweet potato tuber, compound 6 (tR30.2) [Peo-3-O-(6, 6‴-

291

dicaffeoylsoph)-5-O-glc] was found at m/z 1111 and its first fragmentation peak at m/z 949

292

was [Peo-3-O-(6, 6‴- dicaffeoylsoph)]+. Peo-3-O-(6-caffeoylsoph)-5-O-glc (tR30.2) was the

293

7th compound and it showed [M] + ion peak at m/z 949 and the first fragmentation peak at m/z

294

787, [Peo-3-O-(6-caffeoylsoph)]+. Compound 8 at tR 30.2, Peo-3-O-(6-caffeoyl-6‴-p-

295

hydroxybenzoylsoph)-5-O-glc was the major anthocyanin in tuber as well as in leaves. The

296

[M]+ ion peak of this compound was at m/z 1069 and the first fragmentation peak was at 907, 13 ACS Paragon Plus Environment


Page 14 of 34

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[Peo-3-O-(6-caffeoyl-6‴-p-hydroxybenzoylsoph)]+. Peo-3-O-(6-caffeoyl-6‴-feruolylsoph)-

298

5-O-glc was compound 9 at tR 30.2 with its [M]

299

fragmentation

300

hydroxybenzoylsoph)]+. The mass spectral data was compared with those of previous studies

301

of anthocyanins from purple fleshed sweet potato to confirm the position of sugars on

302

aglycon part and the structure of anthocyanins.24 The interesting factor was the structural

303

similarity of the two anthocyanins viz., Cy-3-O-(6-caffeoyl-6‴-p-hydroxybenzoylsoph)-5-O-

304

glc and Peo-3-O-(6-caffeoyl-6‴-p-hydroxybenzoylsoph)-5-O-glc present in both the purple

305

leaves and in the root tubers. These two differ only in their aglycones and this structural

306

similarity might be responsible for the increase in intermolecular self-complexation, which

307

leads to an increase in the intensity of color and stability.25

308

Cytotoxicity study by Hoechst 33342 live cell staining

309

Hoechst staining was used to understand chromatin condensation changes in MCF 10A cells

310

to study the cytotoxicity effects produced by sweet potato root tuber and leaf anthocyanins on

311

a normal cell. At the concentrations used for the study, no changes were noticed in chromatin

312

condensation in the cells as illustrated by fluorescent images (figure 3). This showed that

313

these anthocyanins had no cell toxicity at these concentrations (100-400µg/ml). A previous

314

investigation has compared the anti-proliferative activity of anthocyanins on normal as well

315

as cancer cells and observed that there was an inhibition of cancer cell growth whereas no

316

effect was noticed with normal cells.26

317

Apoptotic studies

318

The protease enzymes, caspases have a leading role in apoptosis; upon activation, caspases

319

cleave multiple proteins leading to apoptosis. A system which uses the FRET principle was

320

used to detect caspase activation in this study. The cell systems were transfected with the

321

plasmid ECFP-DEVD-EYFP. Stable cell lines were generated by using the expression vector

peak

at

m/z

963

+

ion peak at m/z 1125 and the first

representing

[Peo-3-O-(6-caffeoyl-6‴-p-


Page 15 of 34


322

pcDNA3 ECFP-DEVD-EYFP (caspase sensor FRET probe), which was a gift from Prof. J M

323

Tavere and Prof. G. Welsh of University of Bristol, UK. Initially, the cells were transfected

324

with the pcDNA3 ECFP-DEVD–EYFP using lipofectamine LTX (Invitrogen, #15338-100)

325

according to the manufacturer protocol. By selecting the cells in 800 μg/ml of G418

326

(Invitrogen) containing medium for 30–40 days, stably expressing clones were generated.

327

The cells expressing FRET were sorted on the basis of EYFP ﬂuorescence using FACS Aria

328

III. Multiple clones were expanded and the clones that stably maintained homogeneous level

329

of both the probes were used for all the subsequent experiments. The FRET donor was the

330

fluorescent protein ECFP and the acceptor was EYFP linked by the tetrapeptide sequence

331

DEVD. Effector caspases such as caspas-3, upon activation, recognize the tetrapeptide

332

DEVD and cleave it resulting in the loss of FRET in caspase activated cells. Mitochondrial

333

membrane permeability and caspase activation are the signature events of apoptosis, that are

334

absent in necrotic cells. Likewise, apoptotic cells do not exhibit loss of membrane

335

permeability before caspase activation.27 Previous studies of Jeena et.al (2011) have proved

336

the potential of FRET-based approach in real-time detection of caspase activation28. In the

337

present study, fluorescence imaging helped to confirm caspase activation. The anthocyanin

338

concentrations used were in line with previous studies on their antitumor effect on different

339

culture cells.29–32 On the basis of available literature, three different concentrations (100, 200

340

and 400μg/ml) have been selected for apoptotic studies on three cancer cell lines. The

341

differences in the CFP/YFP ratio of the CFP-DEVD-YFP FRET probe due to apoptosis were

342

investigated. Caspase activation was rapid in the stably transfected cells viz., MCF-7, HeLa,

343

and HCT-116 after treatment with 100µg of anthocyanins. Figure 4 demonstrates the effect of

344

anthocyanins on CFP/YFP ratio in MCF-7 cells. Anthocyanins treatment resulted in the

345

breakage of the FRET probe causing an increased CFP/YFP emission ratio, which indicates a

346

a lowering of resonance energy transfer. Similarly, figures 5 and 6 indicate the changes in



Page 16 of 34

347

CFP/YFP ratio in the case of HCT-116 and HeLa cells after the anthocyanin treatment. These

348

results indicated that CFP/YFP ratio in these three cell lines increased with increase in

349

anthocyanins concentration. Anthocyanins from sweet potato leaves as well as root tubers

350

induced apoptosis in MCF-7, HeLa and HCT-116 cells, exhibiting their significant antitumor

351

effect against these cells. The apoptotic effect was relatively larger on MCF-7 cell lines in the

352

case of both anthocyanins (figure 4), indicating that these were more effective against human

353

breast cancer cells at concentrations of 100μg/ml and above. The MCF-7 cells treated with

354

tuber anthocyanins have shown a slightly higher CFP/YFP ratio than that of the cells treated

355

with leaf anthocyanins at all the selected concentrations. At lower concentrations, CFP/YFP

356

ratios of anthocyanins treated HCT-116 cells were similar in case of both tuber and leaves,

357

but at higher concentration the CFP/YFP ratio shows slight increase the cells treated with leaf

358

anthocyanins. The CFP/YFP ratio was comparatively larger in HeLa cells on treatment with

359

leaf anthocyanins at all the selected concentrations. These results indicated the comparative

360

effect of leaf and tuber anthocyanins towards the three selected cancer cell lines. Among

361

these, the tuber anthocyanins have shown a comparatively higher effect of inducing apoptosis

362

on the MCF-7 cell lines, but a slightly higher apoptotic effect was observed in leaf

363

anthocyanins on HCT-116 and HeLa cell lines.

364

Both intrinsic (mitochondrial) and extrinsic (FAS) pathways are responsible for the induction

365

of apoptosis by anthocyanins.33–35 The mitochondrial membrane potential increases on

366

anthocyanin treatment of cancer cells along with the release of cytochrome c in intrinsic

367

pathway. The caspase-dependent anti- and pro-apoptotic proteins were also modulated.

368

However, anthocyanin treatment results in the modulation of FAS and FAS ligand expression

369

in cancer cells in the extrinsic pathway causing apoptosis. Caspase-3 activation and cell death

370

were also caused by cyanidin-3-glucoside and peonidin-3-glucoside.11 Therefore, both these


Page 17 of 34


371

mechanisms might be contributing to the apoptotic effect of anthocyanins from the tuber and

372

leaves.

373

An early study using the anthocyanidins found in the aglycones of majority of anthocyanins

374

in nature showed that they inhibit carcinogenesis by arresting the mitogen-activated protein

375

kinase pathway activation, which might possibly be due to the presence of ortho-

376

dihydroxyphenyl group on their structure.36 Apoptosis was induced in breast cancer cells by

377

the way of caspase-3 activation by the cyanidin and peonidin derivatives of anthocyanins

378

isolated from Oryza sativa L. indica11, which was in accordance with the findings of the

379

current study. As observed in our study, they have also noticed a greater effect with cyanidin

380

glucoside than peonidin glucoside. Purple sweet potato tuber anthocyanins repressed colon

381

and breast cancer cell proliferation depending on time and concentration with 50% inhibitory

382

concentration of approximately 3–7 mg/ml up on treatment for 24 h.37 For a detailed

383

investigation on the effect of anthocyanins against cancer cells, cell cycle analysis has been

384

done.

385

Cell cycle analysis by FACS

386

Since the disruption of cell cycle is very important in the development of cancer, its alteration

387

by phytochemicals is a potential approach in to regulate carcinogenesis.38,39 Anthocyanins

388

have the capability to arrest different stages of cell cycle by affecting cell cycle regulatory

389

proteins such as cyclin A, cyclin D1, p21, p27 and p53 and thereby preventing proliferation

390

of cells. Flow cytometry was used to study the effect of sweet potato leaf and tuber

391

anthocyanins on cell cycle of various cancer cell lines. MCF-7 cancer cells treated with leaf

392

as well as tuber anthocyanins (100μg/ml) for 48 h exhibited a substantial cell cycle arrest and

393

apoptosis induction as evidenced by increased percentage of cells in the sub G0 phase (figure

394

7 a, d and g). The percentage distribution of cells increased only at G0 level when compared

395

to control. The G0 level cell population was 0.40% in the untreated MCF-7 cells (figure 7a), 17 ACS Paragon Plus Environment


Page 18 of 34

396

which increased to 20.5% (figure 7d) and 23.7% (figure 7g) respectively for the cells treated

397

with 100μg/ml of leaf and tuber anthocyanins. In both cases, the distribution percentage of

398

cells in G1, S, and G2/M phases decreased in comparison to control. Similar effect was

399

exhibited by other cell lines also. There was an increase in cells in the sub G0 phase from

400

0.80% for the control to 12.0% and 9.70% respectively, for HCT-116 cells treated with leaf

401

and tuber anthocyanins and no significant increase was observed in the other phases (figure 7

402

b, e and h). According to a previous study, purified cyanidin exhibited superior in vitro anti-

403

proliferative activity in human colon cancer cells (HCT-116) when compared to other

404

anthocyanins.40 Similar results are obtained in the present study also where cyanidin rich leaf

405

anthocyanins exhibited a better effect on HCT-116 cells than peonidin rich tuber

406

anthocyanins. The percentage of HeLa cells increased from 0.3% in the sub G0 phase for the

407

control, to 17.0% and 10.3% respectively for those treated with leaf and tuber anthocyanins

408

(figure 7 c, f and i). The cells present in S phase considerably increased from 15.4% in the

409

control to 21.3% and 21.8% respectively, for anthocyanin treated cells. The S to G2 phase

410

transition was prevented by the accumulated cells in the S phase resulting in a decreased

411

number of tumor cells in G1 phase. A sub-G0 peak also appeared showing the apoptotic effect

412

on different cell lines. Some earlier studies indicated that apoptosis is caused by disruption of

413

the cell cycle. The protein levels of cell cycle related protein such as cyclin-dependent kinase

414

CDK-1, CDK-2, cyclin B1, and cyclin E were down-regulated in peonidin-3-glucoside

415

treatment, whereas CDK-1, CDK-2, cyclin B1, and cyclin D1 were decreased in cyanidin-3-

416

glucoside treatment.11 According to Zakaria et.al (2009), the chemo-preventive property of a

417

chemical is marked by its capacity to cause cell cycle arrest.41 Anthocyanin rich cranberry

418

extract caused a significant arrest of MCF-7 cells in G0/G1 phase and studies on its

419

mechanism revealed that there was direct inhibition of protein expression of CdK4 and cyclin

420

D1 by these anthocyanins whereas indirect inhibition of kinase activity of cylcin D1/CdK4


Page 19 of 34


421

complex in human breast cancer cells. When colon cancer cells were treated with

422

anthocyanins, cell cycle arrest was significantly higher at the G1/G0 and G2/M phases.

423

Progression in cell cycle was controlled by the synergy between cyclin and cyclin-dependent

424

kinases and cyclin kinase inhibitors down-regulate this complex.42 The same mechanism

425

might be acting behind the cell cycle arrest of sweet potato tuber and leaf anthocyanins due to

426

the blockage at G0 phase in these three cancer cells.

427

Effect of structure on anti-proliferative activity of anthocyanins

428

The B ring has only one hydroxyl group in peonidin, while cyanidin has two. An earlier study

429

on the structural relation of bioactivity of anthocyanins reports that at least two hydroxyl

430

groups on the B ring of anthocyanidin is crucial for the activity.43 This information agrees

431

with the present study, in which the cyanidin rich leaf extracts showed a superior effect

432

against colon and cervical cancer cells by inducing apoptosis. There was an exception in the

433

case of breast cancer cells, where tuber anthocyanins showed a slightly greater effect than

434

leaf anthocyanins. The B ring of anthocyanins with hydroxyphenyl structure might be

435

contributing to the activity by suppressing the cell conversion and transactivation of activator

436

protein-1.36 A study with breast cancer induced mice have shown that cyanidin derivates

437

remarkably upheaved the cleavage of caspases-3 through the Bcl-2 regulated apoptotic

438

pathway and behaved as an anti-cancer compound by activating apoptosis.44 Literature

439

showed that phenolic acid derivatives with more hydroxyl groups have better effect against

440

breast cancer than meagerly hydroxylated derivatives. The current study showed that the

441

quantity of caffeic acid rich compound 6 was significantly greater in tuber anthocyanins

442

(20.2%) than that in leaf (3.1%). This result revealed that along with the aglycone part, the

443

presence of acylation also has a decisive role in deciding the anti-proliferative activity of

444

anthocyanins.



Page 20 of 34

445

The present study indicated the potential anti-carcinogenic activity of purple-colored sweet

446

potato tuber and leaf anthocyanins against multiple cancer cell types. These anthocyanins

447

caused cell cycle arrest and consequent inhibition of cancer cell proliferation. Presence of

448

acylated cyanidin and peonidin derivatives in major quantities in the leaves and tubers played

449

a vital role their anti-proliferative effect observed with the three studied cancer cells. The

450

tuber anthocyanins showed comparatively higher effect of inducing apoptosis on human

451

breast cancer cells, while leaf anthocyanins had a slightly greater apoptotic effect on the other

452

two cancer cells. These results suggest the potential of sweet potato anthocyanins in reducing

453

the risk of cancer. These can also serve as natural colorants and open up the possibility of

454

wider application of purple sweet potato tubers and leaves in food industry.

455

Supporting information

456

HR-ESI-MS spectra of nine anthocyanins isolated from purple leaves and root tubers of

457

sweet potato (Figure 1-9).

458 459

ACKNOWLEDGEMENTS

460

The authors acknowledge the financial assistance provided by the Indian Council for

461

Agriculture Research (ICAR), Government of India, through the network project ‘High Value

462

Compounds/Phytochemicals’ for carrying out this study. The analytical support for cell line

463

studies provided by Rajiv Gandhi Centre for Biotechnology, Kerala, Indiais greatly

464

acknowledged.


Page 21 of 34


466

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Konczak-Islam, I.; Yoshimoto, M.; Hou, D. X.; Terahara, N.; Yamakawa, O. Potential



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produced tissue of sweetpotato (Ipomoea batatas L.). J. Agric. Food Chem.2003, 51

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molecular mechanisms: Update from recent decade. Crit. Rev. Food Sci. Nutr.2017, 57

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611 612

Figure 1. HPLC profiles of sweet potato anthocyanins from (a) leaves of Acc. S-1467, (b)

613

root tubers of cv Bhu Krishna, (c) acid hydrolyzed leaf anthocyanins, (d) acid

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hydrolyzed tuber anthocyanins, (e) acid hydrolyzed cyanidin 3-O-glycoside

615

standard and (f) acid hydrolyzed peonidin 3-O-glycoside standard

616



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617 618

619 620

Figure 2. Chemical structure of anthocyanins in the leaves of sweet potato accession S-1467 and root tubers of cv Bhu Krishna

621 622

*caff

= caffeoyl, fer = feruloyl, phb = p-hydroxybenzoyl

623


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624 625

Figure 3. Fluorescent images of MCF-10A cells treated with sweet potato leaf and tuber

626

anthocyanins at different concentrations. (a) control cells, (b-d) cells treated with

627

leaf anthocyanins at 100µg/ml, 200µg/ml and 400µg/ml respectively; (e-g) cells

628

treated with tuber anthocyanins at 100µg/ml, 200 µg/ml and 400 µg/ml

629

respectively (20× magnification).

630

*Cells

marked with white arrowheads indicate nuclear chromatin condensation.

631



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632 633

Figure 4. Fluorescent images of MCF-7 cells treated with sweet potato leaf and tuber

634

anthocyanins at different concentrations. (a) control cells, (b-d) cells treated with leaf

635

anthocyanins at 100µg/ml, 200µg/ml and 400µg/ml respectively; (e-g) cells treated with tuber

636

anthocyanins at 100µg/ml, 200 µg/ml and 400 µg/ml respectively (20×magnification)

637

*Cells

marked with white arrowheads indicate nuclear chromatin condensation

638


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639 640

Figure 5. Fluorescent images of HCT-116 cells treated with leaf and tuber anthocyanins at

641

different concentrations. (a) control cells, (b-d) cells treated with leaf anthocyanins at

642

100µg/ml,200µg/ml and 400µg/ml respectively; (e-g) cells treated with tuber anthocyanins at

643

100µg/ml, 200 µg/ml and 400 µg/ml respectively (20× magnification).

644

*Cells


645



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646 647

Figure 6. Fluorescent images of HeLa cells treated with leaf and tuber anthocyanins at

648

different concentrations. (a) control cells, (b-d) cells treated with leaf anthocyaninsat

649

100µg/ml, 200µg/ml and 400µg/ml respectively; (e-g) cells treated with tuber anthocyanins at

650

100µg/ml, 200 µg/ml and 400 µg/ml respectively (20× magnification).

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*Cells


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654 655

Figure 7. Effect of sweet potato anthocyanins (100µg/ml) on cell cycle of MCF- 7, HCT-116

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and HeLa cells in comparison with their controls. (a) MCF-7 control, (b) HCT-116 control,

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(c) HeLa control, (d-f) MCF-7, HCT-116 and HeLa cells treated with leaf anthocyanins, (g-i)

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MCF-7, HCT-116 and HeLa cells treated with tuber anthocynins

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84x47mm (300 x 300 DPI)

ACS Paragon Plus Environment

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Comparative study on the chemical structure and in vitro anti

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