Differential Partitioning of Triterpenes and Triterpene Esters in Apple

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Differential partitioning of triterpenes and triterpene esters in apple peel Brenton C. Poirier, David A. Buchanan, James Mattheis, and David R Rudell J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b04509 • Publication Date (Web): 22 Jan 2018 Downloaded from http://pubs.acs.org on January 22, 2018

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Journal of Agricultural and Food Chemistry

Differential partitioning of triterpenes and triterpene esters in apple peel

Brenton C. Poirier, David A. Buchanan, David R. Rudell*, Loren A. Honaas, James P. Mattheis Tree Fruit Research Laboratory, USDA-ARS, Wenatchee, Washington 98801 * Author to whom correspondence should be addressed [telephone (509) 664-2280; fax (509) 664-2287; e-mail [email protected]].

1 ACS Paragon Plus Environment


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ABSTRACT:

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Apple peel is a rich source of secondary metabolites, and several studies have outlined the

3

dietary health benefits of ursane-type triterpenes in apple. Changes in triterpene metabolism

4

have also been associated with the development of superficial scald, a postharvest apple peel

5

browning disorder, and postharvest applications of diphenylamine and 1-methylcyclopropene.

6

Previously, studies have generated metabolite profiles for whole apple peel or apple wax. In

7

this study, we report separate metabolic analyses of isolated wax fractions and peel epidermis

8

to investigate the spatial distribution of secondary metabolites in peel.

9

examining previously reported triterpenes, we identified several unreported fatty acid esters of

10

ursane-type triterpenes (C14-C22). All free pentacyclic triterpenes and triterpenic acids, with

11

the exception of β-amyrin, were localized in the wax layer, along with esters of ursolic acid and

12

uvaol. All sterols, sterol derivatives and α-amyrin esters were localized in the dewaxed peel

13

epidermis.

In addition to

14

15

16

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KEYWORDS:

18

apple, Malus, epicuticular wax, triterpene ester, ursenoic acid, 1-methylcyclopropene

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ACS Paragon Plus 2 Environment

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INTRODUCTION

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Apple peel functions as a protective barrier against biotic and abiotic stresses, and preserving

23

the integrity and appearance of peel critical for market success. In apple (Malus pumila), peel

24

epidermal cells and the associated epicuticular wax are a rich source of secondary metabolites;

25

ursolic acid alone accounts for as much as 32-70% of the epicuticular wax layer in fruit of

26

different Malus pumila cultivars1, and 0.15% of peel fresh weight in Malus pumila cv. Delicious2.

27

The composition of apple peel has gained much attention in recent years because of the

28

metabolic changes associated with postharvest storage disorders and the dietary health

29

benefits associated with triterpenes. In addition to potent antioxidant activity, ursane-type

30

triterpenes have also demonstrated potential anti-proliferative activity2-5.

31

Investigation of the triterpene composition of apple peels has led to the identification of

32

a number of novel compounds in the peel of different apple cultivars. There have been

33

mutliple reports of polyhydroxylated oleanolic and ursolic acid derivatives; including the

34

dihydroxy ursanoic acids, corosolic acid2,6 and annurcoic acid4, the trihydroxy ursanoic acids,

35

tormentic acid and euscaphic acid7, and the dihydroxy oleanoic acid maslinic acid2.

36

Additionally, multiple isomers of polyhydroxylated ursanoic acids have been reported that are

37

yet to be fully characterized8.

38

conjugates of ursolic acid (3β-trans-cinnamoyloxy-2α-hydroxyurs-12-en-28-oic acid, 3β-trans-p-

39

coumaroyloxy-2α-hydroxyurs-12-en-28-oic acid, 3β-trans-p-cinnamoyloxy-2α-hydroxyolean-12-

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en-28-oic acid) in apple peel with demonstrated antiproliferative activity2. Uvaol, ursolic acid,

He and Lui (2007) reported cinnamoyl and p-coumaroyl



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corosolic acid, 3β-trans-cinnamoyloxy-2α-hydroxyurs-12-en-28-oic acid, and 3β-trans-p-

42

coumaroyloxy-2α-hydroxyurs-12-en-28-oic acid have also been reported in the wax of apple

43

fruit9.

44

Changes of sesquiterpene and phytosterol metabolism have also been associated with

45

the development of superficial scald10,11. Superficial scald is a postharvest apple peel disorder

46

that occurs in susceptible cultivars after prolonged periods in cold storage, and is characterized

47

by extensive browning of the peel, which does not extend into the interior flesh of the fruit. Its

48

development has been attributed to oxidative damage and can be effectively prevented using

49

the

50

methylcyclopropene (1-MCP)12. Changes in the metabolism of unidentified pentacyclic terpenes

51

within peel precede the onset of superficial scald10. Additionally, applications of DPA and 1-

52

MCP have been shown to alter the metabolism of sterols and sterol conjugates. A 4-fold

53

increase in acyl steryl glucosides and decrease in free sterols were observed prior to the onset

54

of visible superficial scald symptoms (30-90 days) in untreated fruit, whereas the fruit treated

55

with 1-MCP exhibited an increase in in steryl esters11.

antioxidant

diphenylamine

(DPA)

or

the

ethylene

perception

inhibitor

1-

56

Previous studies have focused on the diversity of triterpenes in apple peel or wax

57

separately, without examining the partitioning of metabolites under different conditions. With

58

few exceptions, the surfaces of terrestrial plants are covered by a cuticle comprised of an inner

59

cuticular layer, composed of hydroxy fatty acids crosslinked by esters bonds to create a

60

polyester layer (cutin), the cuticle proper, composed of cutin and intracuticular waxes, and an

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outer epicuticular wax layer of long chain aliphatic compounds and a broad range of cyclic

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compounds13,14. The epicuticular wax layer in apple fruit is a deposition site for secondary


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metabolites including hydroxycinnamic acids and their fatty acid esters15,16 and triterpenes17,18.

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It has also been recently reported that apple wax contains appreciable quantities of C16 – C30

65

fatty acids, including C16:0, C18:0, C18:1, C18:2 C:20, C20:1 and C2219. The interpretation of

66

metabolite profiling experiments would benefit greatly from the added dimension of

67

compound localization. We sought to determine whether triterpene metabolites have discrete

68

localization patterns within the layers of apple peel.

69 70

MATERIALS AND METHODS

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‘Granny Smith’ apples were harvested one month prior to commercial harvest (140 days after

72

full bloom) at a research orchard near Orondo, WA. Assessment of fruit maturity and 1-MCP

73

application was performed after harvest as described previously10. Apples were stored in air at

74

1 °C and removed after four months; four replications (six fruit per replicate) per treatment

75

were used for metabolite extractions.

76 77

Isolation and analysis of peel and wax fractions. Whole apples were submerged in 300 mL of

78

hexanes in an 800 mL beaker and sonicated in a Branson 8510 Ultrasonic Bath (Branson

79

Ultrasonics, Danbury, CT, USA) for 1 min. An additional rinse with hexane was used to collect

80

residual wax from the fruit after removal from the hexane bath. This was repeated 5 times, for

81

a total of six fruit per solvent extraction. The fruit were then subjected to an identical

82

extraction procedure using chloroform as a solvent. Both solvents, containing wax fractions,

83

were evaporated under vacuum at 40 °C using a Bucchi R-200 rotary evaporator. Peel was

84

collected with a potato peeler immediately after chloroform extraction, flash frozen in liquid



85

nitrogen, cryogenically milled to a fine powder, and stored at -80 °C prior to metabolite

86

analysis. Yields were measured for peel (wax removed) and wax fractions and to account for

87

differences in weight between layers in subsequent data processing. Additional metabolite

88

extractions were performed using cortex tissue directly beneath the peel to ensure that

89

compounds identified in epidermal samples were not the result of contamination from cortical

90

cells. Extraction of nonpolar metabolites was performed using 10 mg of each wax fraction, 500

91

mg of peel (wax removed) and 500 mg of cortex tissue as performed previously10,11. Frozen

92

tissue was placed into 2 mL opaque, screw-top microcentrifuge tubes with 100 μL of 0.5 mm

93

(diameter) soda lime glass beads (BioSpec Products, Inc., Bartlesville, OK). 100 μL of α-

94

tocopherol acetate (79.8 ng μL-1) was added as an internal standard, followed by 0.71 mL of 2:1

95

acetone/0.2 M HEPES, pH 7.7. Tubes were centrifuged at 2000g for 15 s before adding another

96

0.71 mL of acetone/HEPES. A MiniBeadbeater (BioSpec Products, Inc.) was used to shake the

97

tubes for 1 min, followed by centrifugation at 16200g for 1min. The supernatant was

98

transferred to a 13 × 100 mm borosilicate test tube (stored in the dark and on ice). Two

99

additional extractions were performed as above using 1 mL of acetone each time. A final

100

extraction was performed with 0.75mL of hexanes, transferring the supernatant to the same

101

test tube. The test tube was vortexed for 15 s and the hexanes phase transferred to a new test

102

tube after phase separation. Two additional phase separations were performed as above,

103

adding 0.75 mL of hexanes to the extract each time and combining the hexanes phases. The

104

hexanes phase was dried under nitrogen, and the extract was re-dissolved in 250 μL of acetone.

105

Samples were filtered through a 0.45 μm PTFE syringe filter prior to analysis.

106


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Liquid chromatography—mass spectrometry. 10 μL of sample were injected into an 1100

108

series (Agilent Technologies) HPLC system operated under the control of Chemstation (B.02.01)

109

using a Chromolith Performance RP-18e (4.6 × 100 mm) monolithic reverse-phase column (EMD

110

Chemicals, Inc., Gibbstown, NJ), a G1315B diode array detector, and a G1946D single-

111

quadrupole mass selective detector with an atmospheric pressure chemical ionization (APCI)

112

source. 80:20 methanol/deionized water (A) and ethyl acetate (B) were used to generate a

113

linear gradient. A mobile phase flow rate of 1.0 mL min-1 was used, and the column

114

temperature was maintained at 20 °C. The mobile phase was initially composed of 100%

115

solvent A for 2 min after sample injection, followed by a linear gradient to increase Solvent B to

116

65% at 21 min, then 100% solvent B was used until 35 min. The DAD was used to recorded

117

spectra (190-700 nm) for the duration of the sample run, followed by MSD analysis. Conditions

118

in the APCI spray chamber were as follows: 414 kPa nebulizer pressure; 425 °C vaporizer

119

temperature; 350 °C drying gas temperature; 4 L min-1drying gas (N2) flow rate; 4 μA and

120

coronal discharge. A fragmentor potential of 170V was used with a capillary potential of 4000

121

V. The MSD was adjusted to monitor positive ions in the scanning mode, continuously

122

monitoring and recording the entire mass spectra within a m/z 100-1200 range.

123

identification

124

chromatographic conditions using an Agilent 6520 Q-TOF/MS controlled by Masshunter

125

(B.05.00) in both negative and positive polarity. APCI spray chamber conditions were as before,

126

but with a fragmentor potential of 125V. Negative ions were monitored in the m/z 100-1200

127

range, and molecular ions (m/z [M-H]-) of triterpene esters were used to confirm identification.

of

fatty

acid

conjugates,

samples

were

128


re-analyzed

with

For

identical


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129

Metabolite Identification and Relative Quantitation. Data acquisition and deconvolution were

130

performed as described previously10. Peak areas were normalized using the α-tocopherol

131

acetate internal standard. To calculate the relative quantities of each metabolite between

132

epidermal and wax layers, the data were further normalized to correct for differences in sample

133

weight used in the nonpolar extraction methods and measured differences in tissue yield (wax

134

and de-waxed peel) per apple. Analytical standards for triterpenes and sterols were purchased

135

from Matreya (State College, PA, USA), Sigma-Aldrich-Fluka (St. Louis, MO, USA), ChromaDex

136

(Irvine, CA, USA); fatty acyl chlorides were obtained from TCI (Portland, OR, USA), Acros

137

Organics (Geel, Belgium), and Sigma-Aldrich-Fluka.

138

metabolites,

139

monogalactosyldiacylglycerols from Avanti Polar Lipids (Alabaster, AL, USA). Steryl esters, steryl

140

glucosides, and acylated steryl glucosides were synthesized as described previously11.

141

Triterpene esters were synthesized using commercially obtained triterpene standards and fatty

142

acyl chlorides. 25 µL of anhydrous pyridine and 500 µL of 0.2% fatty acyl chloride in xylenes

143

were added to a 2 mL borosilicate auto sampler vial containing 0.1 mg of triterpene. Vials were

144

capped and incubated at 130 °C for 4 hours. Vial contents were then transferred to a 15 × 125

145

mm borosilicate test tube and 5 mL of 0.27 M HCL was added to stop the reaction. A phase

146

extraction was performed by adding 2 mL of chloroform and vortexing the tubes for 30 sec.

147

The chloroform phase was transferred to a brown tinted autosampler vial, dried down under a

148

stream of nitrogen, and dissolved in 200 µL of acetone. Peak identification was confirmed by

149

spiking biological extracts with synthesized triterpene ester standards for LC-MS analysis.

150

RESULTS

carotenoid

standards

were

For the identification of cellular

obtained


from

Sigma-Aldrich-Fluka

and

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Isolation and analysis of epicuticular wax and peel epidermis. Malus pumila cv. Granny Smith

152

apples were collected one month prior to commercial harvest; 50% of apples were treated with

153

1µL L-1 1-MCP for 12 h following harvest as performed previously10 and all apples were stored in

154

air at 1⁰C for 4 months prior to metabolite extraction and analysis. Early harvest increases

155

susceptibility to the postharvest storage disorder, superficial scald which is prevented by 1-

156

MCP, making this an ideal model for studying the impact of 1-MCP on peel metabolism. Apple

157

peel epicuticular waxes were isolated and fractionated using hexane and subsequent

158

chloroform extraction to allow for separate metabolic analyses of wax and epidermis.

159

Fractionating wax components based on solubility using a two-solvent isolation procedure

160

allowed us to simplify the sample matrices for more robust LC-MS analysis. Nonpolar

161

metabolites were extracted from wax and epidermal samples and analyzed by LC-MS in positive

162

and negative ion scanning modes, in the m/z 100-1200 range. To ensure that cellular

163

metabolites were not extracted into the wax fractions, authentic standards were used to

164

identify the peaks of metabolites where the tissue-based location is already understood.

165

Relative quantitation of these metabolites from our LC-MS analyses revealed that lipophilic

166

plastidal metabolites (carotenoids chlorophyll a, chlorophyll b, β-carotene, lutein, neoxanthin,

167

and violaxanthin, as well as plastidal lipids (monogalactosyldiacylglycerols) were extracted

168

exclusively from peel epidermis. The same can be said of the plasma membrane lipids,

169

glucosylceramides (Table S1); there was no evidence that cellular integrity was compromised

170

during wax removal. Metabolite data was normalized to account for the differences in yield

171

between wax fractions and de-waxed peel for the purpose of determining metabolite

172

partitioning.



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Apple peel contains several unreported triterpene esters. Our analysis encompassed 42

174

triterpenes and triterpene conjugates (Table 1), with the vast majority displaying discrete

175

localization to either epicuticular wax or peel epidermal cells. Included in our analysis were 18

176

tetracyclic triterpenes, including free sterols (7,9,11), steryl esters (24-26, 28, 30, 32), steryl

177

glucosides (34-36), and acyl steryl glucosides (37-42). Additionally, we identified several

178

pentacyclic ursane-, oleane- and lupane-type triterpenes previously reported in apple peel,

179

including: 3β-hydroxy-lup-20(29)-en-28-oic acid (betulinic acid) (1), 3β-hydroxy-urs-12-en-28-

180

oic acid (ursolic acid) (2), 3β-hydroxy-olean-12-en-28-oic acid (oleanolic acid) (3), 3β-hydroxy-

181

urs-12-en-28-ol (uvaol) (4), 3β-hydroxy-olean-12-ene-28-ol (erythrodiol) (5), 3β-hydroxy-lup-

182

20(29)-ene (lupeol) (6), 3β-hydroxy-olean-12-ene (β-amyrin) (8), and 3β-hydroxy-urs-12-ene (α-

183

amyrin) (10) (Table 1). Peak identifications based on spectra and relative retention times were

184

confirmed using authentic standards. Unfortunately, peaks for oleanolic acid and ursolic acid

185

could not be resolved and appeared as doublet peaks; as was the case with uvaol and

186

erythrodiol. However, uvaol and erythrodiol displayed identical localization patterns and

187

solubility within hexane and chloroform extracts, and we thought it appropriate to quantify

188

them together as a single peak.

189

We also identified 16 fatty acid esters of α-amyrin, uvaol, and ursolic acid that have not

190

been reported in apple fruit (Table 1). Peaks were identified as triterpene esters based on the

191

presence of their triterpene moiety in positive ion mode (m/z [M-(FA+H2O)+H]+), molecular ions

192

(m/z [M-H] -) in negative ion mode when present, and relative hydrophobicity as compared

193

with unmodified triterpenes and previously identified steryl esters11 (Figure S1).

194

assignments were confirmed by spiking biological extracts with triterpene ester standards.


Peak

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195

Standards were synthesized by esterifying fatty acids to the secondary alcohol on triterpene

196

backbones, using fatty acyl chlorides in a nucleophilic addition/elimination reaction20. Fatty

197

acids were attached at the C3 position of amyrins and triterpenic acids at the only available

198

alcohol. No β-amyrin esters were detected, but α-amyrin-3-palmitate (31), -stearate (33), -

199

linoleate (27), and -linolenate (23) were tentatively identified in epidermal metabolite extracts.

200

Ursolic acid-3-stearate (18), -oleate (16), -linoleate (14), -linolenate (12),

201

heneicosylate (21), and -behenate (22) were also identified in wax extracts. Uvaol esters of

202

stearic (19), oleic (17), linoleic (15), and linolenic acids (13) were tentatively identified, but

203

determining the esterification position was not possible due to the presence of two alcohol

204

groups on the triterpene backbone and the lack of regiospecificity of our in-vitro synthesis of

205

triterpene ester standards.

206

Triterpenes and triterpene esters display distinct localization patterns. Previously, application

207

of 1-MCP was shown to induce a dramatic shift in the metabolism of sterol conjugates, leading

208

to an approximate 4-fold-decrease in acyl steryl glucosides and a 2-fold increase in steryl esters

209

4 months after treatment11. We sought to determine whether 1-MCP application also impacts

210

spatial distribution of these metabolites. All phytosterols (including conjugates) localized to the

211

epidermis with >90% specificity and no significant changes in localization were observed

212

following 1-MCP treatment at 4 months. With the exception of β-amyrin, all oleane-, ursane-

213

and lupane-type triterpenes were enriched within the epicuticular wax layer (>90% of total),

214

including: α-amyrin, lupeol, uvaol, erythrodiol, and betulinic acid (Table 2, Figure 1). Due to the

215

abundance of oleanolic and ursolic acids, saturation of the detector response prevented us

216

from performing relative quantitation between wax and epidermal extracts. Esters of


-arachidate (20), -


217

dihydroxy-terpenes and triterpene acids were also localized to the wax layer (>85% of total).

218

Interestingly, the more hydrophobic α-amyrin esters were found exclusively within the

219

epidermis, contrary to the localization of α-amyrin (Figure 1). To ensure that α-amyrin esters

220

were not originating from residual cortex tissue on the epidermal samples, we performed

221

additional extractions and analysis of nonpolar metabolites from the underlying cortex tissue.

222

No appreciable quantities of α-amyrin esters were detected within the cortex as compared with

223

epidermal samples (Figure S2). Although epicuticular waxes are often viewed as a deposition

224

site for hydrophobic secondary metabolites, hydrophobicity alone is a poor determinant of

225

metabolite localization for structurally similar triterpene conjugates in apple peel.

226

DISCUSSION

227

The cyclization of 2,3-oxidosqualene to produce pentacyclic oleane- and ursane-type

228

triterpenes proceeds sequentially through a pentacyclic dammerenyl carbocation, then a

229

lupenyl carbocation before ring expansion gives rise to an oleanyl carbocation, followed by a

230

methyl shift to generate an ursanyl carbocation (Figure 1)21,22. Apples possess at least four

231

multifunctional triterpene synthases that collectively produce oleane, ursane and lupane-type

232

triterpenes. MdOSC1 and MdOSC3 produce lupeol, α-amyrin and β-amyrin21, MdOSC4 produces

233

lupeol, β-amyrin and an additional oleane-type triterpene (germanicol), and MdOSC5 produces

234

lupeol and β-amyrin23. Despite the presence of lupeol synthase activity, very few groups have

235

reported appreciable levels of lupeol or its oxidation product, betulin in apple wax of non-

236

russeted cultivars. However, multiple groups have reported the presence of betulinic acid4,7,8.

237

This contrasts with russeted apple varieties, which contain significant levels of betulin, betulinic


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238

acid, and betulinic acid-3-caffeates24. Esters of lupeol and betulinic acid were synthesized to

239

determine whether these were present in detectable levels in our epicuticular wax fractions,

240

including laurate, myristate, palmitate, stearate, oleate, linoleate, and linolenate. Consistent

241

with previous reports, we did not detect acylated forms of lupeol or betulin in epidermal or wax

242

extracts.

243

To the best of our knowledge, there have been no previous reports of fatty acylated α-

244

amyrins, uvaol or ursolic acid in apple peel. However, similar compounds have been reported in

245

several other species. Fernandes et al. (2013) identified short chain fatty acid esters of α- and

246

β-amyrin within the fruit of Manilkara subsericea25. Fingolo et al. (2013) identified octanoate,

247

decanoate, dodecanoate, tetradecanoate, and hexadecanoate esters of α-amyrin and β-amyrin

248

in extracts of Dorstenia arifolia26. Also reported were the fatty acid conjugates of ursane and

249

oleane-type triterpenes ursa-9(11),12-dien-3-yl decanoate and oleana-9(11),12-dien-3-yl

250

decanoate.

251

regioselectivity in our synthesis of uvaol ester standards eliminated the possibility of further

252

structural elucidation.

253

positions on a broad range of triterpene backbones27,28, and there are likely multiple isoforms

254

of fatty acid conjugates derived from polyhydroxylated triterpenes. To further support this,

255

uvaol-3,28-dimyristate and uvaol-3,28-dipalmitate were identified in metabolite extracts by

256

spiking extracts with the respective diacylated triterpene standards. However, the increased

257

hydrophobicity of these compounds caused them to elute at the very end of chromatographic

258

runs where conditions were not suitable for quantitative analysis.

Although we were able to identify several esters of uvaol, the lack of

Acyltransferases in plants can utilize hydroxyl groups in multiple



Page 14 of 22

259

Application of the ethylene perception inhibitor, 1-MCP is among the most common

260

postharvest application for maintaining the quality of apple fruit during cold storage. Although

261

significant changes in sterol and pentacyclic triterpene metabolism have been documented

262

following 1-MCP treatment, we demonstrate that triterpene partitioning remains unaffected.

263

The consistent localization of sterols and sterol conjugates to the epidermis is not surprising,

264

given the role of phytosterols in regulating membrane dynamics and the numerous reports of

265

sterol conjugates being stored in endosomal compartments, summarized in a recent review29.

266

Significant quantities of oleanolic and ursolic acids were detected in both epidermal and wax

267

extracts, and ursolic acid concentrations of 320-700 mg g-1 have been reported in wax1.

268

Although we were unable to accurately quantify these triterpenic acids, it is clear that a sizeable

269

percentage of each compound is deposited within the epicuticular wax layer (Figure 1). The

270

mechanisms of metabolite partitioning for ursane- and oleane-type triterpenes and the

271

function of this spatial coordination remain to be determined. What we present here is by no

272

means a comprehensive list of the triterpenoids present in apple, but the added information

273

about metabolite localization will aid in the interpretation of other datasets. Changes in

274

epidermal metabolites are more likely to affect cellular dynamics, whereas changes in wax-

275

localized metabolites more likely to alter the physical and chemical characteristics of the fruit

276

surface.

277

ABBREVIATIONS USED

278

1-MCP, 1-methylcyclopropene; APCI, atmospheric pressure chemical ionization; DPA,

279

diphenylamine;

HPLC,

high-performance

liquid

chromatography;


LC-MS,

liquid

Page 15 of 22


280

chromatography–mass spectrometry; MSD, mass selective detector; Q-TOF, Quadrupole time-

281

of-flight.

282

283

Supporting information.

284

Table S1. Localization of epidermal metabolites.; Figure S1. LC-MS identification of triterpene

285

esters.; Figure S2. Nonpolar extracts from peel epidermis and cortex.



REFERENCES 1. Belding, R.D.; Blankenship, S.M.; Young, E.; Leidy, R.B. Composition and variability of epicuticular waxes in apple cultivars. J Amer Soc Hort Sci. 1998, 123, 348-356. 2. He, X.; Liu, R. H. Triterpenoids isolated from apple peels have potent antiproliferative activity and may be partially responsible for apple’s anticancer activity. J. Agric. Food Chem. 2007, 55, 4366– 4370. 3. Eberhardt, M.V.; Lee, C.Y.; Liu, R.H. Antioxidant activity of fresh apples. Nature. 2000, 405, 903-904. 4. Cefarelli, G.; D'Abrosca, B.; Fiorentino, A.; Izzo, A.; Mastellone, C.; Pacifico, S.; Piscopo, V. Free-radicalscavenging and antioxidant activities of secondary metabolites from reddened cv. Annurca apple fruits. J Agric Food Chem. 2006, 54, 803-809. 5. D’Abrosca, B.; Fiorentino, A.; Monaco, P.; Oriano, P.; Pacifico, S. Annurcoic acid: A new antioxidant ursane triterpene from fruits of cv. Annurca apple. Food Chem. 2006, 98, 285-290. 6. Ma, C.M.; Cai, S.Q.; Cui, J.R.; Wang, R.Q.; Tu, P.F.; Hattori, M.; Daneshtalab, M. The cytotoxic activity of ursolic acid derivatives. Eur J Med Chem. 2005, 40, 582-589. 7. He, Q.Q.; Yang, L.; Zhang, J.Y.; Ma, J.N.; Ma, C.M. Chemical constituents of gold-red apple and their αglucosidase inhibitory activities. J Food Sci. 2014, 79, 1970-1983. 8. McGhie, T.K.; Hudault, S.; Lunken, R.C.; Christeller, J.T.; Apple peels, from seven cultivars, have lipase-inhibitory activity and contain numerous ursenoic acids as identified by LC-ESI-QTOF-HRMS. J Agric Food Chem. 2012, 60, 482-491. 9. Szakiel, A.; Pączkowski, C.; Pensec, F.; Bertsch, C. Fruit cuticular waxes as a source of biologically active triterpenoids. Phytochem Rev. 2012, 11, 263-284. 10. Rudell, D.R.; Mattheis, J.P.; Hertog, M.L. Metabolomic change precedes apple superficial scald symptoms. J Agric Food Chem. 2009, 57, 8459-8466. 11. Rudell, D.R.; Buchanan, D.A.; Leisso, R.S.; Whitaker, B.D.; Mattheis, J.P.; Zhu, Y.; Varanasi, V. Ripening, storage temperature, ethylene action, and oxidative stress alter apple peel phytosterol metabolism. Phytochemistry. 2011, 72, 1328-1340.


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12. Zanella, A. Control of apple superficial scald and ripening—a comparison between 1-methylcyclopropene and diphenylamine postharvest treatments, initial low oxygen stress and ultra low oxygen storage. Postharvest Biol. Technol. 2003, 27, 69-78. 13. Chibnall, A.C.; Piper, S.H.; Pollard, A.; Smith, J.A.; Williams, E.F. The wax constituents of the apple cuticle. Biochem J. 1931, 25, 2095-2110. 14. Muller, C.; Reiderer M. Plant surface properties in chemical ecology. J Chem Ecol. 2005, 31, 2621-2650. 15. Riley, R.G.; Kolattukudy, P.E. Evidence for Covalently Attached p-Coumaric Acid and Ferulic Acid in Cutins and Suberins. Plant Physiol. 1975, 56, 650-654. 16. Whitaker, B.D.; Schmidt, W.F.; Kirk, M.C.; Barnes, S. Novel fatty acid esters of p-coumaryl alcohol in epicuticular wax of apple fruit. J Agric Food Chem. 2001, 49, 3787-3792. 17. Belding, R.D.; Sutton T.B.; Blankenship, S.M. Relationship between apple fruit epicuticular wax and growth of Pelaster fructicola and Leptodontidium elatius, two fungi that cause sooty blotch disease. Plant Dis. 2000, 84, 767–772 18. Lara, I.; Belge, B.; Goulao, L.F. A focus on the biosynthesis and composition of cuticle in fruits. J Agric Food Chem. 2015, 63, 4005-4019. Review. 19. Legay, S.; Cocco, E.; André, C.M.; Guignard, C.; Hausman, J.F.; Guerriero, G. Differential lipid composition and gene expression in the semi-russeted "Cox Orange Pippin" apple variety. Front Plant Sci. 2017, 8, 1656. 20. Sonntag, N.O.V. The Reactions of Aliphatic Acid Chlorides. Chem Rev. 1953, 52, 237–416 21. Brendolise, C.; Yauk, Y.K.; Eberhard, E.D.; Wang, M.; Chagne. D.; Andre, C.; Greenwood, D.R.; Beuning, L.L. An unusual plant triterpene synthase with predominant α-amyrin-producing activity identified by characterizing oxidosqualene cyclases from Malus × domestica. FEBS J. 2011, 278, 2485-2499. 22. Xue, Z.; Duan, L.; Liu, D.; Guo, J.; Ge, S.; Dicks, J.; ÓMáille, P.; Osbourn, A.; Qi, X.; Divergent evolution of oxidosqualene cyclases in plants. New Phytol. 2012, 193, 1022-1038. 23. Andre, C.M.; Legay, S.; Deleruelle, A.; Nieuwenhuizen, N.; Punter, M.; Brendolise, C.; Cooney, J.M.; Lateur, M.; Hausman, J.F.; Larondelle, Y.; Laing, W.A. Multifunctional oxidosqualene cyclases and cytochrome P450 involved in the biosynthesis of apple fruit triterpenic acids. New Phytol. 2016, 211, 1279-1294.



24. Andre, C.M.; Larsen, L.; Burgess, E.J.; Jensen, D.J.; Cooney, J.M.; Evers, D.; Zhang, J.; Perry, N.B.; Laing, W.A. Unusual immuno-modulatory triterpene-caffeates in the skins of russeted varieties of apples and pears. J Agric Food Chem. 2013, 61, 2773-2779. 25. Fernandes, C.P.; Corrêa, A.L.; Lobo, J.F.; Caramel, O.P.; de Almeida, F.B.; Castro, E.S.; Souza, K.F.; Burth, P.; Amorim, L.M.; Santos, M.G.; Ferreira, J.L.; Falcão, D.Q.; Carvalho, J.C.; Rocha, L. Triterpene esters and biological activities from edible fruits of Manilkara subsericea (Mart.) Dubard, Sapotaceae. Biomed Res Int. 2013; 2013, 280810. 26. Fingolo, C.E.; Santos, Tde S.; Filho, M.D.; Kaplan, M.A.; Triterpene esters: natural products from Dorstenia arifolia (Moraceae). Molecules. 2013, 18, 4247-4256. 27. Hill, R.A.; Connolly, J.D. Triterpenoids. Nat Prod Rep. 2013, 30, 1028-1065. Review. 28. Chung, I.M.; Siddiqui, N.A.; Kim, S.H.; Nagella, P.; Khan, A.A.; Ali, M.; Ahmad, A.; New constituents triterpene ester and sugar derivatives from Panax ginseng Meyer and their evaluation of antioxidant activities. Saudi Pharm J. 2017, 25, 801-812. 29. Valitova, J.N.; Sulkarnayeva, A.G.; Minibayeva, F.V. Plant Sterols: Diversity, Biosynthesis, and Physiological Functions. Biochem (Mosc). 2016, 81, 819-834.

FIGURE CAPTIONS Figure 1. Partitioning of compounds within triterpene biosynthesis pathways. Hollow arrows denote percentage of compound deposited in wax.1compound not identified in dataset, 2

percent deposition unknown.


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Page 19 of 22


TABLES Table 1. Chromatographic and mass spectral information for identified compounds. Compounds were identified by spiking metabolite extracts with their respective standards. no.

RT (min)

1 2,3 4,5 6 7 8 9 10 11

9.06 9.4/9.52 9.67 16.9 17.83 18.38 18.49 18.66 18.69

12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33

20.13 20.21 20.82 20.9 21.61 22.13 22.67 23.05 23.56 24.05 24.35 26.46 26.48 26.49 26.69 27.93 28.13 28.37 28.73 30.13 30.59 32.76

34 35 36

14.63 15.26 15.61

37 38 39 40 41 42

21.38 21.45 21.85 22.03 22.86 23.53

triterpenes

formula

betulinic acid ursolic/oleanolic acid uvaol/erythrodiol lupeol stigmasterol β-amyrin campesterol α-amyrin β-sitosterol triterpene esters ursolic acid-3-linolenate uvaol linolenate ursolic acid-3-linoleate uvaol linoleate ursolic acid-3-oleate uvaol oleate ursolic acid-3-stearate uvaol stearate ursolic acid-3-arachidate ursolic acid-3-heneicosylate ursolic acid-3-behenate α-amyrin 3-linolenate stigmasteryl linoleate campesteryl linolenate β-sitosteryl linolenate α-amyrin-3-linoleate β-sitosteryl linoleate α-amyrin-3-myristate campesteryl linoleate α-amyrin-3-palmitate β-sitosteryl palmitate α-amyrin-3-stearate steryl glucosides stigmasteryl glucoside campesteryl glucoside β-sitosteryl glucoside acyl steryl glucosides stigmasteryl glucosyl linoleate β-sitosteryl glucosyl linolenate campesteryl glucosyl linoleate β-sitosteryl glucosyl linoleate β-sitosteryl glucosyl palmitate β-sitosteryl glucosyl stearate

C30H48O3 C30H48O3 C30H50O2 C30H50O C29H48O C30H50O C28H48O C30H50O C29H50O C48H76O4 C48H78O3 C48H78O4 C48H80O3 C48H80O4 C48H82O3 C48H82O4 C48H84O3 C50H86O4 C51H88O4 C52H90O4 C48H78O2 C47H80O3 C46H76O2 C47H78O2 C48H80O2 C47H80O2 C44H76O2 C46H78O2 C46H80O2 C45H80O2 C48H84O2 C35H58O6 C34H58O6 C35H60O6 C53H88O7 C53H88O7 C52H88O7 C53H90O7 C51H90O7 C53H94O7

m/z [M-H2O+H]+ calculated experimental

difference (mDa)

439.3577 439.3573 439.3577 439.3575 425.3784 425.3735 409.3835 409.3838 395.3678 395.3675 409.3835 409.3823 383.3678 383.3661 409.3835 409.3821 397.3835 397.3830 m/z [M-(FA+H2O)+H]+ 439.3577 439.3584 425.3784 425.3734 439.3577 439.3584 425.3784 425.3778 439.3577 439.3577 425.3784 425.3784 439.3577 439.3553 425.3784 425.3774 439.3577 439.3583 439.3577 439.3584 439.3577 439.3584 409.3835 409.3831 395.3678 395.3672 383.3678 383.3675 397.3835 397.3855 409.3835 409.3842 397.3835 397.3825 409.3835 409.3840 383.3678 383.3677 409.3835 409.3826 397.3835 397.3827 409.3835 409.3828 m/z [M-(Gluc+H2O) +H]+ 395.3678 395.3661 383.3678 383.3657 397.3835 397.3843 m/z [M-(FA+Gluc+H2O)+H]+ 395.3678 395.3677 397.3835 397.3845 383.3678 383.3662 397.3835 397.3841 397.3835 397.3843 397.3835 397.3843

-0.4 -0.2 -4.9 0.3 -0.3 -1.2 -1.7 -1.4 -0.5


0.7 -5 0.7 -0.6 0 0 -2.4 -1 0.6 0.7 0.7 -0.4 -0.6 -0.3 2 0.7 -1 0.5 -0.1 -0.9 -0.8 -0.7 -1.7 -2.1 0.8 -0.1 1 -1.6 0.6 0.8 0.8

m/z [M-(H2O)2+H]+

m/z [M-H]-

407.364

715.5714 407.367 717.5872 407.367 719.6028 407.365 721.6181 407.367 749.6491 763.6122 777.6788


Page 20 of 22

Table 2. Triterpene partitioning in control and 1-MCP treated fruit at 4 months post-harvest. compound triterpenes (cyclization products) lupeol α-amyrin β-amyrin triterpene esters α-amyrin-3-myristate α-amyrin-3-palmitate α-amyrin-3-stearate α-amyrin-3-linoleate α-amyrin 3-linolenate di-hydroxy triterpenes uvaol/erythrodiol di-hydroxy triterpene esters uvaol stearate uvaol oleate uvaol linoleate uvaol linolenate triterpenic acids betulinic acid triterpenic acid esters ursolic acid-3-stearate ursolic acid-3-oleate ursolic acid-3-linoleate ursolic acid-3-linolenate ursolic acid-3-arachidate ursolic acid-3-heneicosylate ursolic acid-3-behenate free sterols β-sitosterol campesterol stigmasterol steryl esters β-sitosteryl palmitate β-sitosteryl linoleate β-sitosteryl linolenate campesteryl linoleate campesteryl linolenate stigmasteryl linoleate steryl glucosides β-sitosteryl glucoside campesteryl glucoside Stigmasteryl glucoside acyl steryl glucosides β-sitosteryl glucosyl palmitate β-sitosteryl glucosyl stearate β-sitosteryl glucosyl linoleate β-sitosteryl glucosyl linolenate campesteryl glucosyl linoleate stigmasteryl glucosyl linoleate

_______ control ________ % wax % peel t-test

_________1-MCP_______ % wax % peel t-test

91.8 96.5 65.6

8.2 3.5 34.4

0.005 0.001 0.035

80.1 94.5 62.6

19.1 5.5 37.

0.010 0.006 0.004

0 0 0 0 0

100 100 100 100 100

0.004 0.004 0.036 0.011 0.004

0 0 0 0 0

100 100 100 100 100

0.012 0.008 0.047 0.012 0.003

90.9

9.1

3.0E-4

81.7

18.3

0.029

100 86.3 100 100

0 13.7 0 0

0.010 0.040 0.013 0.010

100 96.0 100 100

0 4.0 0 0

0.011 0.013 0.009 0.033

90.1

9.9

4.0E-4

82.5

17.5

0.017

95.6 100 98.5 100 100 100 100

4.4 0 1.5 0 0 0 0

0.015 0.011 0.026 0.057 1.5E-6 0.043 0.001

93.9 97.7 96.2 100 100 100 100

6.1 2.3 3.8 0 0 0 0

0.011 0.001 0.003 0.009 0.008 0.004 0.029

5.1 0 4.0

94.9 100 96.0

0.005 0.002 0.001

3.8 0 92.8

96.2 100 7.2

0.005 0.003 0.007

10.5 9.3 11.1 0 0 0

89.5 90.7 88.9 100 100 100

0.002 0.013 0.020 0.022 0.007 0.017

17.4 5.6 3.8 0 0 0

82.6 94.4 96.2 100 100 100

0.001 0.017 0.027 0.048 0.034 0.026

0 0 0

100 100 100

0.004 0.004 0.016

0 0 0

100 100 100

0.008 0.007 0.005

0 0 0 0 0 0

100 100 100 100 100 100

0.003 0.001 0.002 0.001 0.004 7.0E-4

0 0 0 0 0 0

100 100 100 100 100 100

0.002 0.001 0.002 0.010 0.014 4.0E-4


Page 21 of 22


FIGURES



TOC Graphic


Page 22 of 22

Differential Partitioning of Triterpenes and Triterpene Esters in Apple

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