Bioactive constituents, vitamin analysis, antioxidant capacity and α-glucosidase inhibition of Canna indica L. rhizome extracts

Journal Pre-proof Bioactive constituents, vitamin analysis, antioxidant capacity and α-glucosidase inhibition of Canna indica L. rhizome extracts S. Ayusman, P. Duraivadivel, H.G. Gowtham, S. Sharma, P. Hariprasad PII:

S2212-4292(18)30802-2

DOI:

https://doi.org/10.1016/j.fbio.2020.100544

Reference:

FBIO 100544

To appear in:

Food Bioscience

Received Date: 22 August 2018 Revised Date:

10 February 2020

Accepted Date: 10 February 2020

Please cite this article as: Ayusman S., Duraivadivel P., Gowtham H.G., Sharma S. & Hariprasad P., Bioactive constituents, vitamin analysis, antioxidant capacity and α-glucosidase inhibition of Canna indica L. rhizome extracts, Food Bioscience (2020), doi: https://doi.org/10.1016/j.fbio.2020.100544. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Ltd.

Author Contribution Ayusman Swain: Conceptualization, Methodology, Validation, Investigation, Formal analysis, Funding acquisition, Roles/Writing - original draft, Writing - review & editing. Duraivadivel P.: Methodology, Validation. Gowtham HG: Methodology, Validation. Satyawati Sharma: Supervision. Hariprasad P.: Conceptualization, Resources, Formal Analysis, Project administration, Supervision, Writing - review & editing.

Conflict of interest The authors declare that there is no conflict of interest

1

Bioactive constituents, vitamin analysis, antioxidant capacity and α-glucosidase inhibition

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of Canna indica L. rhizome extracts

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Ayusman S1., Duraivadivel P1., Gowtham H. G2., Sharma S1., and Hariprasad P.1*

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Center for Rural Development and Technology, Indian Institute of Technology Delhi, New Delhi, India 110016 Department of Studies in Biotechnology, University of Mysore, Karnataka, India 570006

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First Author: Dr. Ayusman Swain

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*Address to Whom Correspondence

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Dr. P. Hariprasad

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Assistant Professor

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Centre for Rural Development and Technology,

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Indian Institute of Technology Delhi,

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Hauz Khas, New Delhi, India

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Email: [email protected]

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[email protected]

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Phone : +91 11 2659 1195

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Mobile: +91 8373904447

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Fax: +91 11 2658 2037

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Running Title: Canna indica rhizomes - Bioactive constituents and biological properties

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Abstract

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Rhizomes of Canna indica were studied to determine their potential use as a functional food, a

34

source of vitamins, nutritional and nutraceutical ingredients. Biomass and nutrient

35

characterization showed the rhizomes were a good source of fiber (25.1%), starch (28.5%), crude

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protein (4.72%), and lipids (5.75%) with a total estimated caloric value of 423 Kcal/100 g dry

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weight. The rhizome also had considerable amounts of minerals and vitamins. Acetone extracts

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of rhizomes showed significantly higher antioxidant properties. The IC50 values with DPPH,

39

ABTS+. and O2.- radicals were 21, 23 and 170 µg/ml, respectively. The reducing properties

40

(FRAP and CUPRAC) and DNA protection assay were correlated with the total phenolic and

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flavonoid content of the rhizome extracts. Acetone and methanol extracts showed protection

42

against free radical-induced DNA and protein degradation. In a β-carotene-linoleic acid model,

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the acetone extracts significantly decreased the bleaching of β-carotenoids. In a meat model

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system, the acetone extracts minimized the thiobarbituric acid reactive substance of ground pork

45

meat. α-Glucosidase activity was significantly inhibited using water extracts (IC50 2.35 µg/ml)

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and acetone extracts (IC50 27.1 µg/ml). HR-LCMS/MS analysis of different extracts showed the

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occurrence of different bioactive compounds such as rosmarinic acid, psoromic acid, usnic acid,

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isoeugenitol, ellagic acid, coumaric acid and swietenine. The results suggested that C. indica

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rhizomes might be a potential source of nutrients and metabolites with health benefits.

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Keywords: Canna indica, Rhizome, α-Glucosidase inhibitors. Rosmarinic acid, Psoromic acid,

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Usnic acid.

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1. Introduction

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"Let food be thy medicine and medicine be thy food" is the famous quote of Hippocrates.

56

Ayurveda and traditional medicine systems followed across the world suggest that people

57

consume a nutrient-dense diet to prevent or cure diseases. The traditional Indian system of

58

medicine uses different plants to cure many diseases and disorders. However, the mechanistic

59

basis of their functioning is yet to be studied for several plant species. Canna indica L. (CI)

60

(family: Cannaceae) is a tropical perennial rhizomatous herb. It grows in almost all agro-climatic

61

zones of India and is commonly known as Indian shot or Sarvajaya or Canna lily (Nirmal et al.,

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2007; Van Jaarsveld et al., 2006). Different plant parts of Canna have been consumed from

63

ancient times. The archeological remnants of Canna plants in the regions of Ecuador and Peru

64

indicated its dominance as a staple food in the prehistoric period (Gade, 1966). Tribes of

65

Lepchas, Bhutias and Nepalis on the Indian subcontinent were known to consume the rhizome of

66

CI as food (Mishra et al., 2011). Because of its difficulty in production and processing compared

67

to other plants such as potato, cassava and maize, Canna has been underutilized. In various

68

regions of China and Vietnam, C. edulis is primarily used to extract starch and prepare

69

transparent noodles (Piyachomkwan et al., 2002; Tonwitowat, 1994). Other than starch

70

extraction, utilization of Canna rhizomes as a functional food such as a source of antioxidants,

71

vitamins, minerals, and proteins has not been studied. In folklore medicine, the medicinal values

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of each plant part of Canna have long been documented as a diaphoretic and diuretic with fever,

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with dropsy as a demulcent and to treat suppuration, malaria, diarrhea, rheumatism, dysentery,

74

bursitis and cuts (Duke, 1985; Odugbemi et al., 2008). However, the mechanism and metabolites

75

behind its biological function are yet to be identified.

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Broadly the plant metabolites are categorized into terpenoids, phenolics and alkaloids.

77

Collectively these metabolites improve the fitness of the plant in its natural environment

78

(Harborne, 1998). They are involved in communication, reduction of abiotic stress, protection

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against herbivory and microbial diseases (Akula & Ravishankar, 2011; Sudha & Ravishankar,

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2002). Secondary metabolites of plant origin in crude or purified form are widely used and

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studied for their biological function and application in managing many diseases in humans and

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animals. In recent years the antioxidant potential of secondary metabolites, especially phenolics

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of plant origin are extensively studied for their beneficial effects on humans and animals. Many

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plant metabolites are the natural source of dietary ingredients, which support a healthy life.

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Several natural antioxidants such as chlorogenic acid, caffeic acid, curcumin, gallic acid and

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ferulic acid are reported as compounds of pharmacological importance which are used in drugs

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for curing numerous diseases initiated by excessive reactive oxygen species (ROS) (Suhaj,

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2006). Some plant metabolites have been shown to be lifesaving such as quinine and artemisinin

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against the malarial pathogen (protozoan parasite) (White, 1997); oseltamivir phosphate

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(Tamiflu) to treat influenza virus A and B infection (Ward et al., 2005); morphine used to

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alleviate severe pain (Sneader 1996); apomorphine to treat the "hypomobility" phase of

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Parkinson's disease (FDA, 2004); vinca alkaloids against various types of cancer (Moudi et al.,

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2013) etc. Given the above, the present study aims to evaluate the potential of CI rhizome as a

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source of nutrition, antioxidants and enzyme inhibitors of health importance.

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2. Material and methods

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2.1 Plant material

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The rhizomes of naturally growing CI were collected from the riverbanks of the Cauvery,

99

Mysore, India. The plants were cultivated and maintained at a research field (moist soil and

100

monsoon-influenced humid climate), at the micromodel experimental field, Indian Institute of

101

Technology Delhi, India. The identification of the plant was done at the Botanical Survey of

102

India (BSI), Kolkata, West Bengal, India. The rhizomes were washed in tap water, blot-dried and

103

kept at 4°C for a maximum of 3 days. These rhizomes were used throughout the experiment

104

either as fresh or dried powder for the extraction of metabolites and phytochemical analysis. To

105

dry, the rhizomes were cut with a knife into small pieces of ~0.5 cm3 and dried in a hot air oven

106

(Acumen Labware, Haryana, India) at 45ºC for 4 days. The dried pieces of rhizomes were course

107

powdered in a mixer grinder (Zodiac MG 218 750-Watt, Preethi Kitchen Appliances Pvt. Ltd.,

108

Chennai, India).

109

2.2 Chemicals and reagents

110

The chemicals used in the study were of analytical grade. 2,2-Diphenyl-1-picryl-hydrazyl

111

(DPPH), 2,2-azinobis (3-ethyl benzothiazoline-6-sulfonic acid) diammonium salt (ABTS), 2,2-

112

azobis (2-amidinopropane) dihydrochloride (AAPH), quercetin (QCT), bovine serum albumin

113

(BSA), calf thymus DNA (CT DNA), β-carotene, linoleic acid, thiobarbituric acid (TBA) and

114

1,3,3-tetramethoxypropane and enzymes were purchased from Sigma Chemicals Co. (St. Louis,

115

MO, USA). Gallic acid (GA), butylated hydroxy anisole (BHA), butylated hydroxy toluene

116

(BHT), ascorbic acid and Folin-Ciocalteu reagent were from Hi-Media Laboratories (Mumbai,

117

India). 4-Nitrophenyl α-D-glucopyranoside (PNPG), hydrogen peroxide, 5,5-dithio-bis-(2-

118

nitrobenzoic acid (DTNB), acetylthiocholine iodide (ATCI), 2,4,6-tri(2-pyridyl)-s-triazine

119

(TPTZ) and all other chemicals and solvents were obtained from Sisco Research Laboratory

120

(Mumbai, India).

5

121

2.3 Biomass characterization

122

The proximate analysis was done including total lipid (Soxhlet extract) (AOAC, 2000)

123

and crude fiber (AOAC, 1990) contents. Free starch was determined following an earlier method

124

(Jeong et al., 2010) with some modifications. Briefly, 100 mg dry weight (dw) of powdered

125

rhizome sample was mixed with 3 ml 80% ethanol and sonicated, 150 W (Ultrasonic bath,

126

Guyson International, Leeds, UK) for 10 min at room temperature (30±2ºC). Then the sample

127

was centrifuged (CPR-24 Plus, Remi Laboratory Instruments, Maharashtra, India) at 13000 g for

128

10 min at 4°C. The supernatant was discarded and the process was repeated thrice. The pellet

129

was dried and dissolved in 2 ml distilled water. To this solution, 100 µl 2 M sodium acetate and

130

100 µl 25 U/ml α-amylase solution were added and incubated at 60°C for 2 h. The reducing

131

sugars in the supernatant were separated by centrifuging at 13000 g for 10 min at 4°C and

132

quantified using DNS reagent (Miller, 1959). Briefly, the test solution was mixed with DNS

133

reagent at 1:1 (v/v) ratio in a test tube and placed on the boiling water bath for 15 min. The tubes

134

were cooled to room temperature, 250 µl of solution was transferred to 96 well microtiter plate

135

and absorbance was read at 540 nm (Goncalve et al., 2010). The percentage of free starch was

136

determined using a standard curve of enzymatic starch (corn) hydrolysis.

137

Calorific value (CV) of dried powder of CI rhizome was estimated using a bomb

138

calorimeter (Model RSKT-6, Rico Scientific Industries, New Delhi, India). Briefly, the equation

139

used to calculate the CV is as follows. =



×



− .



ℎ

+



140

Carbon, hydrogen, nitrogen contents of the rhizome were determined using a CHN

141

analyzer (Elementar Analysensysteme GmbH, Euro-EA 3000, Langenselbold, Germany). The

142

samples were weighed (5 mg) in small tin containers in triplicate. The containers were closed 6

143

and folded over the edge and placed in the elemental analyzer. Different elements were measured

144

as a percentage of initial starting weight using the elemental analyzer software. The crude protein

145

content in the rhizome was estimated by using a Kjeldahl conversion factor of 6.26 (Tucker &

146

Debusk, 1981). Quantitative analysis of inorganic elements (essential metals) such as Na, K, Fe,

147

Ca, Mg, Mn, Cu, Co and Zn were determined using inductively coupled plasma mass

148

spectrometry (Agilent 7900 ICP-MS, Agilent Technologies, Santa Clara, CA, USA). The

149

powdered sample was digested with HNO3 (ultra-pure grade) using closed-vessel microwave

150

digestion (Titan MPS microwave sample preparation system, PerkinElmer, Inc., Waltham, MA,

151

USA). Commercially available standard solution (ICP multi-element standard solution XXI for

152

MS, Merck, Darmstadt, Germany) was used to prepare the calibration curves.

153

2.4 Analysis of vitamins

154

2.4.1 Preparation of samples

155

Vitamins (water and fat-soluble) in the dried rhizome was determined using HPLC and

156

LC-MS/MS. For HPLC, standard solutions of vitamins, A (retinoic acid), D (cholecalciferol and

157

cholecalcitriol), and E (α-tocopherol and α-tocopheryl acetate) were prepared in methanol and

158

(L-ascorbic acid) was prepared in water. Similarly, the rhizome powders (mortar ground) were

159

directly extracted with methanol and water. Vitamin standards and rhizome extracts (RE) were

160

run in the HPLC system with an RP C18 column (Agilent-1260, Agilent Technologies, Santa

161

Clara CA, USA). The specific HPLC conditions are provided in Table 1.

162

LC-MS/MS analysis was done in a Waters Acquity PDA system with an Accucore RP

163

C18 150 x 2.1 mm, 2.6 µm column with an auto-sampler (Waters UPLC-TQD, Waters India Pvt.

164

Ltd., Bangalore, India). The crude methanol and hexane extracts reconstituted with methanol and

165

were tested for fat-soluble vitamins (A, D and E). The water extract was tested for water-soluble

7

166

vitamins (B-complex and C). A gradient solvent system with (A) 0.01% TFA in water (pH 3.9)

167

and (B) methanol was applied. The gradient profile started with 95:5 (A:B) and remained

168

constant for the first 4 min then went to 2:98 (A:B) for the next 6 min. It was constant for the

169

next 3 min and then 0:100 (A:B) for the next 17 min (Klejdus et al., 2004). The flow rate was

170

maintained at 0.7 ml/min and the column temperature was kept at 30°C. The analytical

171

wavelength was set at 280 nm and MS/MS analysis was done in both positive and negative

172

mode. Ionisation method: Atmospheric pressure ionization and the mass range was 50-1000 m/z.

173

The results were analyzed using the mass bank database (https://massbank.eu/MassBank) by

174

checking the base peaks and other major fragment ion peaks with the existing mass spectra data

175

of standard vitamins.

176

2.5 Phytochemical extraction and analysis

177

Soxhlet extraction of crude metabolites was performed using dried rhizome powder in

178

solvents with increasing polarity (hexane
179

The extracts were concentrated under low pressure using a rotary evaporator (Buchi R-205,

180

Fisher Scientific, Reinach, Switzerland). Stock solutions (mg/ml) of RE were prepared by

181

dissolving the dried samples in a common solvent, dimethyl sulphoxide (DMSO), whereas the

182

water extract was dissolved in water. Presence of alkaloids, terpenoids, flavonoids, tannins,

183

phlobatanins, saponins, steroids and cardiac glycosides in rhizome was analyzed following the

184

methods described earlier (Mujeeb et al., 2014) with little modification (Supplementary Table 1),

185

and the results were expressed as present (+) and absent (-) for tests.

186

2.6 Total phenol and flavonoids

187

Total phenolic contents in different extracts were quantified using Folin-Ciocalteu (FC)

188

reagent. Briefly, 250 µl sample solution was added to 1 ml diluted (1:9) FC reagent. After 5 min

8

189

incubation, 750 µl 1% Na2CO3 solution was added. Then the sample was incubated at 30℃ for 2

190

h and absorbance was measured at 760 nm (Slinkard & Singleton, 1977). The total phenolic

191

contents of solvent extracts were expressed as equivalents of gallic acid (µg GAE/mg crude

192

extract).

193

Total flavonoid content was quantified by mixing sample solution with 2% AlCl3 in

194

methanol in 1:1 ratio. After 15 min incubation at 30℃, the sample and blank absorbance were

195

read at 415 nm. The absorbance of the blank was subtracted from that of the sample. The total

196

flavonoid content was expressed as equivalents of quercetin (µg QCTE/mg crude extract) (Berk

197

et al., 2011).

198

2.7 Total antioxidant activity (phosphomolybdenum method)

199

RE were mixed with reagent solution (0.6 M sulfuric acid, 28 mM sodium phosphate and

200

4 mM ammonium molybdate) at a 1:10 ratio, and incubated for 90 min at 95°C. The absorbance

201

of the mixture was measured at 695 nm. The total antioxidant capacity of RE was expressed as

202

equivalents of ascorbic acid (µg AE/mg crude extract) (Berk et al., 2011).

203

2.8 Radical scavenging activity

204

2.8.1 DPPH radical scavenging

205

RE (10 µl) were mixed with 250 µl DPPH solutions (0.2 mM in methanol) in a 96 well

206

microtiter plate (WMP). After incubating the samples for 15 min at 30℃ in the dark, the

207

absorbance was read at 517 nm (Blois, 1958). DPPH radical scavenging capability of RE was

208

calculated and expressed as percent inhibition using the following equation: % =

!" #

− !" $ !" #

× 100

209

The IC50 values of crude extracts were compared qualitative with the standards (GA, BHA and

210

BHT). 9

211

2.8.2. ABTS cation radical scavenging

212

ABTS solution was prepared by mixing 2.45 mM potassium persulfate and ABTS (7

213

mM) in water and allowing the mixture to stand for 15-16 h in the dark at 30℃). The ABTS

214

solution was diluted 10 times with methanol before use. RE (10 µl) was added to 200 µl of

215

ABTS solution in a 96 WMP. After incubating the samples for 30 min at 30℃, the absorbance

216

was read at 734 nm (Re et al., 1999). ABTS cation radical scavenging activity of RE was

217

compared with the standards (GA, BHA and BHT) and represented as IC50.

218

2.8.3 Superoxide anion (O2−) radical scavenging

219

To the 175 µl reagent mixture (10 µl 0.1 mg/ml riboflavin, 10 µl 12 mM EDTA

220

ethylenediaminetetraacetic acid, 5 µl 1 mg/ml nitroblue tetrazolium (NBT), 100 µl 50 mM

221

potassium phosphate buffer (KPB, pH 7.8) and 50 µl methionine), 25 µl RE were added into a 96

222

WMP. The reaction mixture was incubated at 30℃ for 10 min using a 20 W fluorescence lamp

223

(DLF Phase-3, Philips, Gurugram, Haryana, India) at 0.5 meter distance. The absorbance of the

224

sample was read at 560 nm and superoxide anion (O2−) radical scavenging activity of RE was

225

expressed as IC50 and compared with the standard (BHA) (Dasgupta & De, 2004; Martinez et al.,

226

2001).

227

2.9 Reducing Power

228

2.9.1 Cupric ion reducing antioxidant capacity (CUPRAC) assay

229

To 150 µl of premixed reaction mixture containing 50 µl 10 mM CuCl2, 50 µl 7.5 mM

230

neocuproine in 95% ethanol and 50 µl 1 M ammonium acetate buffer (pH 7.0) and 25 µl RE

231

were added into a 96 WMP. Similarly, the blank was without CuCl2. The mixture was incubated

232

for 30 min at 30℃, and the absorbance of the sample, BHA as a positive control and blank were

233

read at 450 nm (Apak et al., 2006). The results were expressed as µg BHAE/mg of RE.

10

234

2.9.2 Ferric ion reducing antioxidant power (FRAP) assay

235

RE (10 µl) were added to 240 µl of FRAP reagent containing 20 mM TPTZ ligand in 80

236

mM HCl, 20 mM FeCl3 and 0.3 M acetate buffer (pH 3.6) at a 1:1:10 ratio in a 96 WMP and

237

incubated for 30 min at 37℃. The sample absorbance was read at 593 nm and compared with the

238

standard, which was prepared by taking a different concentration of FeSO4 with 20 mM TPTZ

239

and 0.3 M acetate buffer (pH 3.6) at a 1:1:10 ratio (Oyaizu, 1986). The results were expressed as

240

µg GAE/mg of RE.

241

2.10 Metal chelating activity

242

RE (200 µl) were mixed with 10 µl 2 mM FeCl2 solution into a 96 WMP and incubated

243

for 5 min. Ferrozine (20 µl, 5 mM) was added to initiate the reaction and a similar reaction

244

without ferrozine served as a control. The sample and blank absorbance were read at 562 nm

245

after 10 min incubation at 30°C (Aktumsek et al., 2013). The metal chelating activity was

246

expressed as µg EDTAE/mg of RE.

247

2.11 Biomolecular protection assay

248

2.11.1 DNA protection assay

249

The protective effect of RE against hydroxyl radical-induced metal-assisted DNA

250

degradation was studied using CT DNA. RE (10 µl) were added to 10 µl TE buffer (pH 8) (10

251

mM Tris-HCl and 1 mM EDTA) containing 280 ng of DNA and incubated for 5 min. To the

252

mixture, Fenton’s reagent (8 µl) was added and incubated for 2.5 h at 30℃ (Karas et al., 2013).

253

The integrity of DNA was analyzed using electrophoresis on 1% agarose gel (Agarose

254

Electrophoresis system, Thermo Fisher Scientific, Mumbai, India) followed by ethidium bromide

255

staining (0.5 µg/ml) for 15 min. The DNA degradation pattern was observed with UV

11

256

illumination (UVstar, Biometra GmbH, Gottingen, Germany). The DNA sample without any

257

extract and Fenton's reagent was used as a control.

258

2.11.2 Anti-protein oxidation assay

259

BSA (0.5 mg) was dissolved in phosphate buffer (pH 7.3) and incubated with peroxyl

260

radicals generating AAPH (2,2-azobis (2-methylpropio-namidine) dihydrochloride) (50 mM)

261

with or without different concentrations of RE. After 2 h incubation at 30℃, the integrity of

262

protein samples was analyzed using SDS-PAGE. The proteins bands were visualized by staining

263

the gel with 0.2% Coomassie Brilliant Blue R-250 in methanol:water:glacial acetic acid (5:5:1)

264

overnight and then destaining (same as staining solution but without the Coomassie Brilliant

265

Blue R-250 dye) until clear bands of protein were observed (Mayo et al., 2003).

266

2.11.3 β-carotene linoleic acid model

267

β-carotene (2 ml, 0.5 mg/ml in chloroform) was mixed with 40 mg linoleic acid and 400

268

mg Tween 40. The solution was dried under low pressure in a rotary evaporator at 40°C.

269

Distilled water (50 ml) was added and the flask was vigorously shaken. The reaction was

270

initiated by adding 3.5 ml of the above solution to 500 µl acetone extract (100 and 200 ppm of

271

GA equivalent phenol) in a test tube. After the addition, the samples were measured 470 nm at 0

272

min. The reaction mixtures were incubated in a water bath at 50°C. The samples was measured

273

every 15 min for 120 min. BHA was used as the standard for comparative analysis. A control

274

without RE or standard was also used. Blank samples were prepared by mixing all the samples

275

and reagents except β-carotene. Blank absorbance was subtracted from that of the sample to get

276

the corrected absorbance. The inhibition % was determined using the following equation (Oh &

277

Shahidi, 2018).

278

Antioxidant activity (%) = [1− (S0−St)/(C0−Ct)] × 100

12

279

(S0: Absorbance of the test sample at 0 min, St: Absorbance of the test sample at every 15 min,

280

C0: Absorbance of control at 0 min and Ct: Absorbance of control at every 15 min)

281

2.11.4 Meat model system (TBARS value)

282

The boneless pork meat was purchased from a local market in New Delhi, India. Pork

283

meat (40 g) was pasted in a pre-chilled mortar and pestle, added to 10 ml Millipore water (Elix 3

284

UV Water Purification System (120 V/60 Hz), Millipore, Merck Life Science, Mumbai, India)

285

and the test sample (10 ml acetone extract containing 150 and 300 ppm of GA equivalent phenol)

286

or the positive control (10 ml, 300 ppm BHT). A blank was also prepared without any

287

antioxidants. The samples were mixed and heated at 80°C in a water bath for 40 min with

288

frequent stirring. The mixture was allowed to cool to room temperature and the contents were

289

again pasted. The contents were stored in plastic bags for 7 days at 4°C (Oh & Shahidi, 2018)

290

and analyzed at 532 nm for their oxidative state using the TBARS test (Shahidi & Hong, 1991)

291

on 0, 3, 5, and 7 days.

292

Trichloroacetic acid (10%, w/v, 3 ml) was added to meat samples (1 g) in centrifuge

293

tubes and Vortexed (Remi Cyclo, CM-101 Plus, Mumbai, India) for 2 min. TBA reagent (0.02

294

M, 3 ml) was added and Vortexed again for 30 s. After centrifugation at 3000g for 10 min, the

295

supernatants were filtered through a Whatman No. 3 filter paper. The samples were kept in a

296

water bath at 95°C for 45 min, then cooled to room temperature. The absorbance of pink TBA-

297

MDA adducts was read at 532 nm. TBARS values were calculated using a standard curve,

298

plotted using 1,1,3,3-tetramethoxypropane (a precursor of MDA). TBARS values were

299

calculated for RE and the results were expressed as mg MDA eq/kg of sample.

300

2.12 Enzyme inhibitory assays

301

2.12.1 α-Amylase inhibition

13

302

RE (200 µl) were mixed with 200 µl α-amylase (2 U/ml) prepared in phosphate buffer

303

(pH 6.9) and incubated for 10 min. Further, 200 µl 1% starch (potato) solution was added and the

304

mixture was incubated for 30 min at 30°C. A blank was maintained with all the reagents except

305

RE to check 100% enzyme activity. The reaction was terminated by the addition of 200 µl DNS

306

reagent (12 g sodium potassium tartrate tetrahydrate in 8.0 ml 2 M NaOH and 20 ml 95 mM 3,5-

307

dinitrosalicylic acid solution) and was boiled for 10 min in a water bath at 85–90 °C. The

308

mixture was cooled to room temperature and was diluted with 5 ml distilled water, and the

309

absorbance was read at 540 nm. The α-amylase inhibitory activity of RE was expressed in

310

percentage (Zengin et al., 2014).

311

2.12.2 α-Glucosidase inhibition

312

RE (50 µl) were mixed with 50 µl 1 U/ml α-glucosidase in phosphate buffer (pH 6.8) and

313

incubated for 10 min in a 96 WMP. Then 50 µl 3 mM glutathione (reduced) and 50 µl 10 mM

314

PNPG were added and incubated for 20 min at 35°C. One control was prepared by mixing all

315

reagents except RE to check the maximum released product. Another blank was prepared by RE

316

and adding all other reagents excluding enzyme. The reaction was terminated by adding 50 µl

317

0.2 M sodium carbonate solution. The sample and blank absorbance were read at 400 nm. The

318

inhibitory activity was expressed in percentage (Zengin et al., 2014).

319

2.12.3 Anticholinesterase activity assay

320

Acetylcholinesterase

(AChE)

inhibitory

activity

of

RE

were

carried

out

321

spectrophotometrically (Ellman et al., 1961). To 150 µl 0.1 M PB, 20 µl RE and 20 µl 1 U/ml

322

enzyme solution were added and mixed in a 96 WMP and incubated for 10 min at 30℃ followed

323

by the addition of 15 µl 10 mM DTNB. Then 15 µl 14 mM ATCI was added to initiate the

14

324

reaction and further incubated for 20 min at 30°C. The absorbance of the reaction mixture was

325

read at 412 nm and the AChE inhibitory activity was expressed in percentage.

326

2.13 HR-LCMS analysis of rhizomes extracts

327

HR-LCMS analysis of ethyl acetate, acetone, methanol and water extracts were done

328

using a 6200 series Q-TOF (Q-Exactive Plus Biopharma-High Resolution MS, Thermo Fischer

329

Scientific, Waltham, MA, USA) mass spectrometer coupled to HPLC equipped with a UV–Vis

330

detector. A 0.2 ml/min flow rate was used with injection volume 5 µl; ESI parameters: both

331

negative and positive ion mode; mass range 100–1200 m/z. The solvent system: (A) formic acid

332

(0.1%, v/v) and 10 mM ammonium formate and (B) acetonitrile + 0.1% formic acid. Gradient

333

mobile phase (solvent A:B): (i) 65:35, from 0 to 0.5 min, (ii) 45:55, from 10 min (iii) 5:95, from

334

25 to 33 min (iv) 65:35, at 35–40 min of total run time.

335

2.14 Statistical analysis

336

The results from the experiments were statistically analyzed using the one way analysis

337

of variance (ANOVA) with the Statistical Package for the Social Science v. 16 software (SPSS

338

Inc., Chicago, IL., USA). The significant difference between the means was compared using the

339

highest significant difference (HSD) as obtained using Tukey's test at the p≤0.05 level.

340 341

3. Results and discussion

342

3.1 Proximate and quantitative analysis

343

The results of biomass characterization and nutrient analysis of CI rhizome is shown in

344

Table 2. The rhizome of CI was found to be a source of energy-dense food which had ~423

345

kcal/100 g dw energy, which ranks it ahead of cassava, wheat, rice, corn and sorghum

346

(Montagnac et al., 2009). The presence of a desirable amount of fibers, starch, protein, lipid,

15

347

minerals and vitamins leads to the benefit of consuming the rhizome. Dietary fiber consists of

348

remnants of plant cells resistant to hydrolysis (digestion) by the alimentary enzymes of humans.

349

The rhizome contains 25% crude fiber, which was less than early reports of 33.1% in CI

350

(Okonwu & Ariaga, 2016), close to the white type ginger (21.9%) (Ajayi et al., 2013) and higher

351

than cassava and potato. Canna spp. are known to produce round to oval-shaped starch granules

352

as large as 100 µm (average 30 - 70 µm), which is larger than cassava starch (12 - 15 µm) and is

353

mainly used to prepare noodles or as ingredient in wheat noodles (Wandee et al., 2015). The

354

study rhizome of CI had 28% of starch which is significantly lower in comparison to C. edulis

355

(48.9%) (Piyachomkwan et al., 2002). The lipid content in rhizome was found to be 5.75% on a

356

dw basis (Table 2). This is relatively higher than a report of Okonwu and Ariaga (2016), where

357

they reported 4.35% in CI rhizome, whereas it was significantly higher than cassava roots

358

(0.28%) (Montagnac et al., 2009). The crude protein content was found to be 4.7% on a dw basis

359

which is higher than C. edulis (3.26%) and cassava (1.15%) (Piyachomkwan et al., 2002). On the

360

other hand, the roots of the ginger were reported to have about 12.1% protein (Ajayi et al., 2013).

361

The higher ash content (11.5%) was consistent with the minerals determined separately, which is

362

comparable to different varieties of C. edulis (5.11 – 5.56%) (Piyachomkwan et al., 2002) and

363

ginger rhizome (4.95 – 7.45%). ICP-MS analysis was used to measure the potential of CI

364

rhizome as a source of minerals (Table 2). Among all tested elements, K was found to be most

365

abundant followed by Na, Mg, and P, and a considerable amount of Ca, Mn, Fe, Cu and Zn were

366

also found in the rhizome. RE were also found to have a number of secondary metabolites such

367

as alkaloids, saponin tannin, flavonoids and phenolics, which were only determined qualitatively

368

(Supplementary Table 2).

16

369

Vitamins are the chemical compounds not synthesized in humans and animals. Hence, a

370

plant diet is considered as a primary source of vitamins. Because of their redox chemistry, their

371

role as an enzyme cofactor or their antioxidant activity, they are beneficial for humans. The

372

occurrence of these vitamins in the rhizome of CI has not generally been reported. Ong and

373

Siemonsama (1996) reported the presence of 0.1 mg vitamin B1 and 10 mg vitamin C in 100 g

374

fw (fresh weight) of CI rhizome. The results of this study are shown in Table 2. Vitamin A

375

(retinoic acid) was probably present in both the cis and trans-retinoic acid forms (Supplementary

376

Fig 1). The peak at Rt 14.39 min shows a fragmentation pattern where the peaks of m/z 301.2

377

(100%), 283, 205, 131 matches with that of retinoic acid in LC-MS/MS analysis (Kane et al.,

378

2008). Although HPLC chromatogram supported the presence of vitamin E (Supplementary Fig

379

2), but it was not detected with LC-MS/MS analysis. Several base peaks were observed in the

380

negative mode LC-MS/MS chromatogram (Supplementary Fig 3), which may be attributed to the

381

metabolic products of α-tocopherol such as γ-CMHHC, γ-CDMOHC, carboxyl δ-tocopherol, δ-

382

CDMOHC and α-CMHHC

383

(Supplementary Fig 4) and quantified to be 11.0 µg/g fw. The HR-LCMS of ethyl acetate and

384

acetone extracts showed the presence of pantothenic acid (vitamin B5) (Table 7). The above

385

results showed the possible utilization of CI rhizome as a vitamin source. Also, the results

386

obtained are consistence with reports of 5-35 µg/g wt vitamin A in cassava roots and, 0.107 and

387

0.296 mg/100 g pantothenic acid in cassava and potato, respectively (Bradbury, 1988; Woot-

388

Tsuen, & Jardin, 1968). Whereas among these three, vitamin C was the highest in cassava (20.6

389

mg/100 g) (Montagnac et al., 2009). Vitamin D was not detected in the rhizome of CI, and other

390

vitamins such in the B-complex were not analyzed.

391

3.2 Antioxidant and biomolecule protection activity

(Zhao et al., 2010). Vitamin C was found with HPLC

17

The antioxidant activity measured with crude extracts of CI rhizome is shown in Table 3.

392 393

The IC

394

ABTS and O2.− radical scavenging activity compared to other solvent extracts tested. In the case

395

of DPPH and ABTS assay, the standard, GA showed the lowest IC50. Whereas, the IC50 values of

396

BHA was comparable to acetone extract in the O2.− radical scavenging assay (Table 3).

50

values measured using acetone extract was significantly (p≤0.05) lower for DPPH,

397

In both the CUPRAC and FRAP assays, acetone extracts showed significant (p<0.05)

398

higher reducing power (Table 3). In CUPRAC assay, the reducing power of acetone extracts

399

were not significantly different from methanol extracts but higher than other extracts. Similar

400

activities were shown by solvent extracts for the FRAP assay. Acetone extracts showed

401

significantly (p≤0.05) higher reducing power in comparison to other extracts (Table 3).

402

The metal ion chelating power of different extracts against ferrous ion are shown in Table

403

3 as an equivalent of EDTA/mg of extract. Methanol extracts shown the highest chelating

404

activity followed by acetone and ethyl acetate. Results in Table 3 show that among all solvent

405

extracts, acetone extracts have the highest total antioxidant capacity followed by methanol and

406

ethyl acetate extracts. Water extracts showed the least activity.

407

The etiology of numerous human and animal diseases is linked to the free radicals

408

generated with stress causing damage to cellular constituents. Even though all living organisms

409

have multiple defense mechanisms to tackle the adverse effect of reactive oxygen species (ROS)

410

generated in the body, but the reduction/failure of these mechanisms leads to diseases conditions.

411

To prevent this condition, it is recommended to consume a diet rich in antioxidants (Prior & Cao,

412

2000). Usually, plants have an array of antioxidant systems because they have to cope with a

413

wide range of biotic and abiotic stresses throughout their life. The antioxidant capacity of plants

414

is related to their total phenolic or total flavonoid contents (Kähkönen et al., 2001; Miliauskas et

18

415

al., 2004; Singleton et al., 1999). Prior and Cao (2000) established a linear correlation between

416

the total phenolic content of wines and their antioxidant properties. Also, the disease protection

417

properties of wines were linked to their free radical scavenging and the transition metal chelating

418

capabilities of flavonoids fractions. Consistent with the previous reports, acetone extracts which

419

showed the highest antioxidant activity, also showed the highest total phenolics and flavonoids,

420

in comparison with other extracts (Table 3).

421

Mishra et al. (2012) found that diethyl ether, ethyl acetate and acetone extracts of C.

422

edulis alone or in combination showed the highest DPPH radical scavenging activity. Further

423

higher concentrations of flavonol and total proanthocyanidins were studied in these fractions.

424

Extracts of CI aerial plant parts had antioxidant properties (Joshi et al., 2009) and

425

hepatoprotective effects (Kaldhone, 2009). In similar studies, the authors correlated this

426

observation with the antioxidant properties of the extracts. Further, these correlations were

427

extended to macromolecular protection (DNA, protein and lipid) from these plant extracts. LC-

428

MS analysis of CI RE obtained a wide range of metabolites which were previously reported to be

429

antioxidant (Table 7).

430

The capability of RE to protect DNA or protein is shown in Fig 1(a). Acetone extracts

431

were found effective in protecting DNA damage at lower concentrations. Methanol was next

432

followed by ethyl acetate and water (little protection). Acetone extracts which showed higher

433

antioxidant and metal chelating activity, also showed higher DNA protection ability and are

434

correlated with metal ion chelation and toxic radical scavenging activity. Similar results were

435

seen using the protein protection assay where acetone extracts protected BSA from damage

436

caused by the AAPH radical at lower concentrations (Fig 1(b)) followed by methanol. Extracts

437

of ethyl acetate and water showed partial protection with the highest concentration used.

19

438

The β-carotene bleaching assay is based on the degradation of β-carotene entrapped in

439

linoleic acid micelles due to the effect of hydroperoxides and free radicals generated with the

440

heat-induced oxidation of linoleic acid. Plant extracts with potential antioxidants have been

441

reported to prevent the damage of β-carotene by scavenging the free radicals (Oh & Shahidi,

442

2018). Acetone extracts of CI rhizome were found to protect the β-carotene from the damaging

443

effect of hydroperoxides and free radicals up to 82% in comparison to control (11%) and BHA

444

(88%) over 120 min incubation period. Also, the β-carotene protection was correlated with the

445

phenolic content of RE (Table 4).

446

Ground pork meat without antioxidant amendment had the highest MDA. Whereas

447

acetone extracts of CI rhizome and BHT decreased the level of MDA significantly (Table 5).

448

3.3 Enzyme inhibitory activity

449

Diabetes Mellitus (DM) is a metabolic disorder of glucose metabolism of multiple

450

etiologies. The condition is characterized by chronic hyperglycemia resulting from decreased

451

insulin secretion or altered response of cells to insulin action, or both. One of the effective

452

measures adopted is to manage the excess postprandial rise of blood glucose level. It is mainly

453

achieved through the intake of proper diet or by using α-amylase and α-glucosidase inhibitors

454

(Tadera et al., 2006). However, long-term use of drugs (Acarbose, Miglitol, Metformin, Sulfonyl

455

Urea, etc.) is often reported to adversely affect human health (Fujisawa et al., 2005; Kelble,

456

2005; Lebovitz, 1997). Alternatively, several natural molecules of plant origin which are

457

consumed by humans reversibly inhibit the activity of α-amylase and α-glucosidase, thereby

458

reducing the rate of free sugar release. Polyphenolic fractions from plants such as catechin

459

gallates, quercetin, isoquercetin and rutin have been studied for their α-amylase and α-

460

glucosidase inhibiting properties (Li et al., 2009). A significant α-glucosidase inhibitory activity

20

461

was shown by CI water extracts and acetone extracts (Table 6). The enzyme inhibition plots for

462

acetone and water extracts (Fig. 2 (a) and (b)) indicated that the rhizome is rich in secondary

463

metabolite(s) as natural α-glucosidase inhibitors. It was also observed that acetone and ethyl

464

acetate extracts inhibit the enzyme activity of α-amylase up to 21 and 18%, respectively, at 166

465

µg/ml and beyond, which was also about the concentration where the solubility of the extracts

466

was decreased. Other extracts were shown to have no inhibitory activity against the enzyme even

467

at the highest concentration used. This suggested the presence of bioactive compounds that can

468

control the activity of enzymes related to the release of free sugar molecule from oligomer or

469

polymers. On the other hand, Purintrapiban (2006) observed that the aqueous extracts of CI roots

470

which had higher total phenolics compounds (catechin) induced 2-deoxy-(3H) glucose uptake in

471

cultured L8 muscle cells and was correlated with increased glucose transporter isoforms 1 and 4

472

on the cell surface.

473

Tripathi (2004) reported the inhibition of AChE activity by extracts of Punica granatum

474

bark or CI root, along with other plant-derived molluscicides in the nervous tissue of Lymnaea

475

acuminate. However, with in vitro conditions, a significant reduction in AChE was observed. In

476

this study, none of the RE showed AChE inhibition.

477

3.4 HR-LC MS/MS analysis of RE

478

Although the crude extracts of different plant parts of Canna were reported to have some

479

biological activity, any effort to correlate the activity with the phenolic metabolites has been

480

minimal. Previously, Sook Yun (2004) reported the presence of 4 phenylpropanoids such as

481

caffeic acid, rosmarinic acid, caffeoyl-1-4-hydroxyphenyllactic acid and salvianolic acid B from

482

the rhizome of Canna edulis. HR-LC MS/MS analysis of different RE of CI showed a wide range

483

of metabolites with reported diverse biological activities (Table 7). The antioxidant,

21

484

macromolecular protection and enzyme inhibitory activities shown by these extracts may be

485

because of the synergistic effect of these metabolites. The presence of relatively higher

486

concentration of metabolites such as usnic acid, ellagic acid, p-coumaric acid, rosmarinic acid,

487

psoromic acid, phenylacetic acid and swietenine like compound in the rhizome indicated its

488

potential use as an antioxidant, anti-inflammatory or anticancerous and to cure/manage diseases

489

related to heart, dementia, etc. The involvement of some unidentified metabolites in the above-

490

studied activities cannot be ignored. In most cases, swietenine was reported in the seeds of

491

Swietenia sp. which were studied for their antidiabetic properties (Dewanjee et al., 2009). The

492

presence of swietenine or swietenine like compounds in canna rhizome has a potential benefit as

493

DM medication. Many naturally occurring phenolic compounds and flavonoids such as

494

quercetin, taxifolin, luteolin, curcumin, p-coumaric acid, caffeic acid, resveratrol, and many

495

other polyphenolics have been studied for their significant α-glucosidase inhibitory activities

496

(Jiang et al., 2017; Proença et al., 2017; Rasouli et al., 2017). Thus the phenolic compounds

497

(Table 7) in acetone extracts of CI such as psoromic acid, usnic acid and rosmarinic acid could

498

be potential inhibitors of α-glucosidase. Further study on individual compounds may show the

499

enzyme-ligand interaction and the importance of CI rhizome as a valuable source of drug

500

ingredients for DM.

501 502

4. Conclusions

503

The rhizomes of CI are good source of nutrients, i.e., starch, dietary fibers, vitamins and

504

minerals, and are comparable to C. edulis and different varieties of ginger. Antioxidant activity

505

of rhizome extracts indicated their potential use in preventing oxidative damage in food and

22

506

biological systems. Further, α-glucosidase inhibitory properties of RE and other metabolites that

507

may have health benefits suggested its potential use as a functional food for diabetic patients.

508 509

Acknowledgments

510

We are grateful to the Council of Scientific and Industrial Research (CSIR), New Delhi

511

for financial support and our premier research institute, the Indian Institute of Technology Delhi

512

(IITD) for providing research space and necessary facilities. We are thankful to the Nanoscale

513

Research Facility (NRF), IITD, Sophisticated Analytical Instrument Facility (SAIF), IIT

514

Bombay and SAIF, the Central Drug Research Institute (CDRI) Lucknow for providing HPLC

515

and LC-MS/MS facilities.

516 517

Conflict of interest

518

The authors declare that there is no conflict of interest

519 520

Human and animal rights

521

This article does not report any studies with human or animal subjects.

522 523

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Table 1. Extraction and HPLC conditions for different vitamins

Sl Vitamin Extraction

A

Dried rhizome in methanol

2

D

Dried rhizome in methanol and hexane*

3

E

1

4

C

Dried rhizome in hexane and petroether*

Fresh rhizome in water

Mobile Phase A: Methanol B: 20 mm PB (pH 2.5) A:B ratio 90:10 Run time: 10 min Flow rate: 1.0 ml/min Methanol Run time: 10 min. Flow rate: 1.0 ml/min A: Methanol B: Water A:B ratio 96:4 Run time: 10 min Flow rate: 1.0 ml/min 1 mM NaH2PO4, 1 mM EDTA, pH adjusted to 3.0 by Phosphoric acid Run time 5 min. Flow rate: 1.0 ml/min

Column Temperat ure

Injection Volume

Wavelength (nm)

Reference

30℃

20 µl

325 and 350

Cosmosil technical note

30℃

15 µl

265

Cosmosil technical note

45℃

50 µl

292

Gimeno et al. (2000)

30℃

25 µl

245

Campos et al. (2009)

*Both solvent extracts were separately analyzed for vitamins

1

Table 2. Proximate, ultimate, vitamin, and mineral composition of Canna indica rhizome Proximate composition (100 g dry weight) Crude Fiber (wt%, extractives-free basis) Starch (%) Total Crude Protein (wt%, extractives-free basis) Total Lipid (%) Calorific values (bomb calorimeter) (kcal/100 g dw) Ultimate analysis (Atomic wt%, dry-ash free basis) Nitrogen Carbon Hydrogen Minerals (mg/100 g dry biomass basis) Na Mg P K Ca Mn Fe Cu Zn Vitamins (µg/g dry biomass basis) Vitamin A (Retinoic acid)** Vitamin D (D3 and D2) # Vitamin E (Tocopherol)$ Vitamin B5 (Pantothenic acid)* Vitamin C (L-Ascorbic acid)∆**

25±2 28±3 4.7±0.6 5.7±1.2 423

0.75 40.4 6.41

30.7 13.2 6.8 199 2.13 0.04 0.53 0.04 0.25

10.3 ---11

∆

µg/g Fresh biomass basis, *Detected using HR-LCMS, **Detected in both HPLC and LCMS, Only its metabolic products were detected in HR-LCMS, #Not detected in either HPLC or LCMS. $

2

Table 3. Total phenolic, flavonoids, and antioxidant activities of different extracts and standards Rhizome extracts and standards Ethyl acetate Acetone Methanol Water GA BHA BHT

Total Phenolics$

Total Flavonoids∆

DPPH*

ABTS*

230±10c

91±4c

48±4e

53±10f

334±10a 250±10b 110±10d ND ND

220±10a 122±4b 0 ND ND ND

21±3c 23±3d d 34±2 42±3e 61±4f 114±5g a 2.8±0.8 4.3±0.9a 7.2±1.8b 12±1b 16±3c 19±1c

Superoxide* CUPRAC#

FRAP$

Metal chelating@

Total Antioxidant activityΨ

NA

230±10b

91±10c

1.3±0.2c

300±10

170±10a 210±10b NA ND 164±10a ND

640±10a 590±10a 50±4c ND ND

180±10a 122±10b 50±2d ND ND

2.9±0.2a 4.1±0.4b NA ND ND ND

520±10a 350±10b 80±10d ND ND ND

*IC50 in µg/ml; #µg BHAE/mg extract; $µg GAE/mg extract; @µg EDTAE/mg extract; ∆µg QCTE/mg extract; Ψµg AE/mg extract; ND-Not determined, NA: No activity showed. Results are mean values of three determinations ± SD. Means sharing the same superscript are not significantly (p≤0.05) different from one another.

3

Table 4. Inhibitory effect of acetone extract of C. indica and standard (BHA) against βcarotene oxidation in a β-carotene-linoleate model system at 50°C for 120 min. Sample Control (no antioxidant) Acetone extract (200 ppm)* Acetone extract (100 ppm)* BHA (200 ppm)

% Inhibition after 120 min 11±1c 82±4a 56±4b 88±3a

Results are mean values of three determinations ± SD. Means sharing the same superscript are not significantly (p≤0.05) different from one another.

4

Table 5. TBARS values (mg MDA eq/Kg) in a meat model system over a 7-day period in the presence of an acetone extract of C. indica and standard (BHT). Sample Control Acetone extract (300 ppm)* Acetone extract (150 ppm)* BHT (300 ppm)

Day 0 1.2±0.0d

Storage Period Day 3 Day 5 6.5±0.2c 6.9±0.2d

Day 7 8.7±0.3c

0.081±0.01a

0.65±0.01a

0.96±0.01a

1.2±0.1a

0.45±0.02c

2.9±0.1b

3.6±0.1c

4.5±0.1b

0.13±0.01b

0.55±0.02a

2.5±0.1b

4.3±0.1b

Results are mean values of three determinations ± SD. Means sharing the same superscript are not significantly (p≤0.05) different from one another. *The ppm values in parenthesis for rhizome extract indicates GA equivalent phenol.

5

Table 6. Enzyme inhibitory activity of different solvent extracts of Canna indica rhizome Rhizome extracts

IC50 for Enzyme Inhibition Experiments α-amylase α-glucosidase Acetylcholinesterase

Ethyl acetate

21% (166)

N

N

Acetone Methanol Water

18% (166) N N

27±3 N 2.4±0.3

N N N

N – no inhibition, Values in the parentheses represents the maximum concentration (µg/ml) of solvent extract used.

6

Table 7. List of major secondary metabolites identified using HR-LCMS/MS in solvent extracts of Canna indica rhizome. (The ethyl acetate, acetone, methanol and water solvent extracts were separately subjected to HR-LCMS/MS.) Sl.

Secondary Metabolites

Rt (min)

1

m-Salicylic acid*

7.48

2

Phenyl acetic acid*

8.03

3

Ellagic acid*

8.94

4

p-Coumaric acid*

9.15

5

4-Hydroxystyrene*

9.16

6

Triamcinolone*

7

Rosmarinic acid*

8

(-)-Usnic acid*

9

Coumarin*

10 Isoeugenitol*

11 Pantothenic acid ** 12

Acetylsalicylic acid ** (aspirin)

9.29

Biological Importance Ethyl acetate extract Analgesic, Antiinflammatory, Anti-pyretic Rab-prenylation inhibitor, Antioxidant Antioxidant’ Antiproliferative, Apoptosis-inducing Antioxidant, Antiinflammatory, Antibacterial, Analgesic Anti-angiogenic, Antiimflammatory Self-regulating drug carriers

Antioxidant, Dioxygenase inhibitor Antioxidative, 10.80 Cardiovascular-protective’ Antibiotic Antibacterial, Antiinflammatory, 11.63 Anticoagulant, Antifungal, antiviral, Antitumor Antioxidant, Antiinflammatory, 12.80 Cardiovascular properties, Anesthetic, Analgesic Vitamin B5, Synthesis of 5.57 Coenzyme A 10.01

10.07 Anti-inflammatory drug

Reference(s)

Pierpoint (1994) Deraeve et al. (2012) Landete (2011) Lou et al. (2012); Pei et al. (2016) Yue et al. (2015) Cato et al. (2001); Cevc & Blume (2003); FughBerman & Ernst (2001) Lee & Scagel (2009); Petersen (2003) Behera, Mahadik, & Morey (2012); Ingólfsdóttir (2002); Verma & Sharma (2012) Cai et al. (2004); Huang, Cai, & Zhang (2009); Yasunaka et al. (2005)

Daniels et al. (2011) Bean & Hodges (1954); Lipmann et al. (1947) Berk et al. (2013); Zucker & Peterson (1970)

Acetone extract 1

m-Hydroxyphenylpyruvic acid*

8.06

2

Psoromic acid*

8.79

3

Ellagic acid*

8.92

Possible antioxidant Rab-prenylation inhibitor, Antioxidant Antioxidant’

-Deraeve et al. (2012) Landete (2011) 7

4

5 6 7

8 9

1 2 3 4 5

p-Coumaric acid*

9.16

Antiproliferative, Apoptosis-inducing Antioxidant, Antiinflammatory, Antibacterial, Analgesic

oHydroxyphenylpyruvic 10.01 Possible antioxidant acid lactone* Antioxidant, Dioxygenase Rosmarinic acid* 10.03 inhibitor Antioxidative, Usnic acid* 10.81 Cardiovascular-protective, Antibiotic Acetylsalicylic acid * * 9.91 Anti-inflammatory (aspirin) Stimulates mitochondrial Lipoamide ** 14.31 biogenesis Methanol extract m-HydroxyphenylPhenolic acid; Possible 8.10 pyruvic acid* antioxidant. Rab-Prenylation Inhibitor, Psoromic acid* 8.83 Antioxidative o-Hydroxyphenyl10.04 Possible antioxidant. pyruvic acid lactone* Antioxidant, Dioxygenase Rosmarinic acid* 10.05 inhibitor Antioxidative, Usnic acid* 10.83 Cardiovascular-protective, Antibiotic

6

Pseudohypericin*

Antiretroviral activity, 11.27 Anticancer property, Apoptosis inducing

7

3-(4-ydroxyphenyl) pyruvic acid **

9.87

1

3,4-Dihydroxyphenylpropionic acid*

6.73

Antioxidant property

2

Choline **

1.64

Essential nutrient

3

Metaraminol **

2.69

Adrenergic receptor agonist

Phenolic acid; Possible antioxidant Water extract

Lou et al. (2012); Pei, Ou, Huang, & Ou (2016)

-Lee & Scagel (2009); Petersen (2003) Behera et al. (2012); Ingólfsdóttir (2002); Verma & Sharma (2012) Berk et al. (2013); Zucker & Peterson (1970) Shen et al. (2011)

Deraeve et al. (2012) -Lee & Scagel (2009); Petersen (2003) Behera et al. (2012); Ingólfsdóttir (2002); Verma & Sharma (2012) Çırak, Radušienė, & Çamas (2008); Meruelo, Lavie, & Lavie (1988); Schempp, Simon-Haarhaus, & Simon (2002) --

Tang et al. (2011) Hollenbeck (2012); Fischer et al. (2010); Lee et al. (2010) McDonald & Santucci (2004) 8

4

Fendiline **

5

Swietenine **

7.16

Antianginal agent Anti-Hyperglycemic 12.82 property

Bayer & Mannhold (1987) Dewanjee et al. (2009)

* HR LCMS in negative mode ** HR LCMS in positive mode

9

Fig 1. Visualization of the damage induced by hydroxyl radicals on genomic DNA (a) and AAPH on protein (BSA) (b) in the presence and absence of acetone extracts from C. indica using agarose gel electrophoresis and SDS PAGE analysis. (a) Lane 1. DNA incubated without Fenton’s reagent; Lane 2. DNA incubated with Fenton’s reagent; Lanes 3-12, DNA incubated with Fenton’s reagent in the presence of 0.04, 0.08, 0.17, 0.35, 0.70, 1.39, 2.79, 5.58, 11.1 and 22.3 µg/ml of acetone extract, respectively (final concentrations). (b) Lane 1: BSA incubated without AAPH. Lane 2: BSA with AAPH, Lane 3 to 9: BSA with AAPH in presence of 2.6, 5.2, 10.4, 20.8, 41.6, 83.2 and 166 µg/ml, of acetone extract, respectively.

Fig 2. α-Glucosidase inhibition plot for acetone extract (a) and water extract (b) of C. indica with increasing concentrations (mean values).

Fig 1. (a) and (b)

Fig 2. (a) and (b)

Highlights Canna indica was found to be a good source of starch, vitamins and minerals. Rhizomes were shown to be a good source of antioxidants, showing significant activity in food and biological model systems. Rhizome extracts were found to contain α-glucosidase inhibitory metabolites. Major compounds such as rosmarinic acid, psoromic acid, usnic acid, ellagic acid, coumaric acid, isoeugenitol and swietenine were identified using HR LC-MS/MS.