Effects of Applied Nitrogen Amounts on the Functional Components of

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Effects of Applied Nitrogen Amounts on the Functional Components of Mulberry (Morus alba L.) Leaves Mari Sugiyama, Makoto Takahashi, Takuya Katsube, Akio Koyama, and Hiroyuki Itamura J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b01922 • Publication Date (Web): 31 Aug 2016 Downloaded from http://pubs.acs.org on September 2, 2016

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Journal of Agricultural and Food Chemistry

1: Effects of Applied Nitrogen Amounts on the Functional Components of Mulberry (Morus alba L.) Leaves

Mari Sugiyama,*,† Makoto Takahashi,†,‡ Takuya Katsube,§ Akio Koyama,ǁ,¶ Hiroyuki Itamura┴

†

Resource and Environment Research Division, Shimane Agricultural Technology Center,

2440 Ashiwata-cho, Izumo, Shimane 693-0035, Japan ‡

Department of Agriculture, Forestry, and Fishery, Shimane Prefectural Government, 1

Tonomachi, Matsue, Shimane 690-8501, Japan §

Department of Biological Applications, Shimane Institute for Industrial Technology, 1

Hokuryo-cho, Matsue, Shimane 690-0816, Japan ¶

National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki 305-8602,

Japan ǁ

Urasoe Silver Human Resources Center, 1-7-2 Inanse, Urasoe, Okinawa 901-2128, Japan

┴

Faculty of Life and Environmental Science, Shimane University, 1060 Nishikawatsu-cho,

Matsue, Shimane 690-8504, Japan

*Corresponding

author

(Tel:

+81-853-22-6804;

Fax:

[email protected])

ACS Paragon Plus Environment

+81-853-21-8380;

E-mail:


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Abstract

2

Our study investigated the effects of applied nitrogen amounts on specific functional

3

components in mulberry (Morus alba L.) leaves. The relationships between mineral elements

4

and the functional components in mulberry leaves were examined using mulberry trees

5

cultivated in different soil conditions in four cultured fields. Then, the relationships between

6

the nitrogen levels and the leaf functional components were studied by culturing mulberry in

7

plastic pots and experimental fields. In the common cultured fields, total nitrogen was

8

negatively correlated with the chlorogenic acid content (R2 = −0.48) and positively correlated

9

with the 1-deoxynojirimycin content (R2 = 0.60). Additionally, differences in nitrogen

10

fertilizer application levels impacted each functional component in mulberry leaves. For

11

instance, with increased nitrogen levels, the chlorogenic acid and flavonol contents

12

significantly decreased, but the 1-deoxynojirimycin content significantly increased. The

13

selection of the optimal nitrogen application level is necessary to obtain the desired functional

14

components from mulberry leaves.

15 16

Keywords: functional component; chlorogenic acid; flavonol; 1-deoxynojirimycin; mulberry

17

leaf; nitrogen application

18


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Introduction

20

Mulberry (Morus spp.) trees have traditionally been cultivated as silkworm feed. In

21

addition, the market demand for their use in health foods is increasing. The benefits of

22

mulberry leaf’ functional components have been reported in various countries

23

example, 1-deoxynojirimycin (DNJ), inhibits the elevation of blood glucose levels. As an

24

α-glucosidase inhibitor, DNJ suppresses the absorption of glucose in the intestines 2. Whereas,

25

flavonols in mulberry leaves have high anti-oxidative effects, as well as inhibitory activities

26

against hypertension, arteriosclerosis, and the development of cancer cells

27

3-(6-malonyglucoside) is the major flavonol glycoside component in mulberry leaves 9, and it

28

enables blood glucose adjustments because of its suppressive effect on oxidative stress in the

29

liver. Although both quercetin 3-(6-malonyglucoside) and DNJ suppress blood glucose

30

elevation, their mechanisms are different

31

polyphenol in mulberry leaves, also plays an important role in providing anti-oxidative

32

effects.

1-6

. For

5,7,8

. Quercetin

10

. Chlorogenic acid, an abundantly contained

33

In raw material production, quality control, as well as technological developments to

34

increase yields, are important. Generally, the contents of the crop’s functional components are

35

influenced by cultivation conditions, such as the variety planted, period of cultivation, growth

36

stages, environmental conditions, and fertilizers applied. Our previous study showed that the

37

flavonol contents of mulberry leaves differ depending on the variety

38

radiation influences flavonol and DNJ contents in mulberry leaves 12. Other researchers 3,13-15

39

also reported that the DNJ contents in mulberry leaves differ depending on the variety.

40

Kimura et al. 3 and Nakanishi et al.

41

is highest in young leaves. Constantinides et al. 17 stated that the DNJ contents were different,

42

depending on the cultivation location. Mudau et al.

16

11

, and that the solar

reported that the DNJ content increased in August and

19

reported that the polyphenolic content



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43

of bush tea increased after nitrogen applications, but Stewart et al.

reported that the

44

flavonol levels of plant tissues were higher after lower nitrogen applications. Matsunaga et al.

45

21

46

applied nitrogen. Thus, we hypothesized that the amount of applied nitrogen also influences

47

the flavonol contents of mulberry. Among various fertilizers, nitrogen is known to influence

48

the yield of mulberry leaves 18, therefore, investigating the nitrogen levels is important.

reported that the catechin content of tea plants increased with decreasing amounts of

49

The purpose of the present study was to examine the effects of nitrogen application

50

levels on chlorogenic acid, flavonol, and DNJ, which are important functional components in

51

mulberry leaves, with the goal of achieving the maximum yields of both mulberry leaves and

52

the targeted functional component. First, the relationships among mineral elements and

53

functional components were examined using mulberry trees in common cultured fields in

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four different locations. Second, the relationships between the nitrogen levels and the

55

functional components was further studied using mulberry trees in plastic pots and in an

56

experimental field.

57 58

Materials and Methods

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Field Study

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Mulberry trees of ‘Ichinose’ (Morus alba L.) were cultivated at the following four

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common cultured fields in Shimane Prefecture, Japan, in 2007: Field A: Onuki, Sakurae-cho,

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Gotsu; Field B: Shikaga, Sakurae-cho, Gotsu; Field C: Onan-cho, Ochi; and Field D:

63

Kisuki-cho, Unnan. Mulberry trees were cultivated for tea leaves in Fields A and B, for

64

silkworm feed to produce materials for Chinese medicine in Field C, and for silkworm feed to

65

produce silk in Field D. These four common fields with different soil conditions were selected

66

to investigate whether field locations affected the functional components in mulberry leaves.


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Although the end-purposes of the mulberry leaves were different, all four of the fields were

68

cultivated using the method for silkworm feed production and the climatic conditions were

69

almost the same.

70

The conditions at the locations and soil textures in each common cultured field were as

71

follows: Field A: Located beside the Gonokawa River, with sandy soil covered by

72

accumulated humus. The soil had limited water- and fertilizer-holding capabilities, and was

73

highly permeable; Field B: Located beside the Gonokawa River, like Field A, but the sandy

74

soil did not have an accumulation of humus; Field C: Located in the mountainside, with clay

75

soil that included plenty of gravel because the land was prepared by excavating the mountain;

76

and Field D: Located in the mountainside, with sandy loam that included plenty of humus on

77

the mild mountain-slope behind an individual’s home. The fertilization of the four fields is

78

shown in Table 1. The soil sampling examinations were conducted on June 7th in Field D and

79

June 14th in Fields A, B, and C. Soil samples were collected in each field at a distance of 30

80

cm from the mulberry stock.

81

The mulberry leaves were harvested on September 13th in Field D, and September 14th

82

in Fields A, B, and C. Two leaves, which were located at positions one-third from the branch

83

top, were sampled from one branch per stock of normal growth (including 20 stocks in Fields

84

A and D, and 10 stocks in Fields B and C). The tree ages in our study were 17 years old in

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Field C and 15 years old in Field D. The tree ages of Fields A and B were unknown.

86 87

Pot experiment

88

The effects of the nitrogen application levels on the functional components in mulberry

89

leaves were investigated using plastic pots at the Experiment Field of Shimane Agricultural

90

Technology Center (Izumo, Shimane, Japan) in 2010. The mulberry cultivar used in this study



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was ‘Ichinose’. On April 6th, 2010, the 2-year-old nursery stocks were planted in 25-L plastic

92

pots containing decomposed granite soil and bark manure at a 1:1 ratio. The mulberry cultivar

93

branches were trimmed, leaving only one or two new shoot branches. Five experimental

94

groups (Groups I, II, III, IV, and V; eight pots per group) were defined by the amount of

95

ammonium sulfate fertilizer applied to investigate the effects of nitrogen. On April 14th, June

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9th, and July 2nd, 2010, ammonium sulfate fertilizer was applied to provide nitrogen at I: 1 g,

97

II: 2.5 g, III: 5 g, IV: 10 g, and V: 15 g per pot. On April 14th, magnesium multi-phosphate

98

and potassium sulfate fertilizers were also applied to provide phosphorous and potassium at

99

0.32 g and 1.1 g per pot, respectively. On July 26th, 2010, completely opened leaflets of the

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youngest leaves and the subsequent two leaves on the same branch were sampled.

101 102

Field experiment

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The cultured field experiment was conducted at the Experimental Field of Shimane

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Agricultural Technology Center. On April 18th, 2010, 1-year-old nursery stocks of mulberry

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cultivar ‘Ichinose’ were planted in a 150 cm wide furrow with 50 cm spacing intervals

106

between trees. After planting, cultivation and fertilization were performed according to the

107

established method for 1 year. Four experimental groups were defined based on the amount of

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ammonium sulfate applied to provide nitrogen: O: 0 kg, I: 6 kg, II: 15 kg, and III: 30 kg per

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1,000 m2 in 2011 and 2012. The four groups were replicated twice in the same field. The

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yearly ammonium sulfate amount was divided into two, and half was applied in 2011 on April

111

5th and the remainder on July 15th, and in 2012 on April 9th and July 21st. The basal fertilizer

112

included magnesium multi-phosphate and potassium sulfate, which provided phosphorous and

113

potassium at 3.2 kg and 7.4 kg per 1,000 m2, respectively. Before bud break in spring, the

114

mulberry trees were pruned at the branch’ bases, and then the re-grown branches were


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harvested in 2011on July 11th and October 4th, and in 2012 on July 9th and September 19th.

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Mulberry tree growth and yield evaluations, and mulberry leaf sampling, were conducted on

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the day of, or prior to, harvesting. Leaf color was evaluated using a chlorophyll meter (SPAD

118

502, Minolta Camera, Co., Ltd., Osaka, Japan). The youngest opened leaves with completely

119

opened leaflets, and the subsequent two leaves of two branches with moderate growth, were

120

sampled from each stock. For each group, 20 stocks (duplicating 10 stocks per group) were

121

used in the investigation.

122 123

Soil analyses

124

Air-dried soil was passed through a 2-mm mesh sieve. Soil pH and electro-conductivity

125

values were measured based on the air-dried soil (weight) and deionized water (volume) at a

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1:2.5 ratio. Total carbon and nitrogen contents of the soil were measured by the dry

127

combustion method using a CN Corder (SUMIGRAPH NC-900, Sumika Chemical Analysis

128

Service, Ltd., Osaka, Japan). The cation exchange capacity was measured using the

129

semi-micro Schollenberger method, and available phosphate acid was measured by the Truog

130

method. Cation exchangers were detected as follows: potassium was measured by flame

131

photometry, and calcium and magnesium were measured by the atomic absorption method.

132 133

Extraction procedures and mineral elements analysis in mulberry leaves

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Sampled leaves were dried in a convection oven (MOV-212F, Sanyo Electric, Inc., Osaka,

135

Japan) at 60°C for 36–48 h. Dry matter samples were ground into a fine powder using a food

136

mill (IFG-700G, Iwatani, Inc., Tokyo, Japan) and then used for the quantitative determination

137

of functional components and mineral elements. Dry mulberry leaf powder (100 mg) was

138

suspended in 10 mL 60% ethanol aqueous (v/v) and stirred for 3 h at 30°C in an incubator



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(LTI-600 SD, Tokyo Rikakikai, Inc., Tokyo, Japan). After centrifugation at 10,000 ×g for 5

140

min, the extracted solution was filtered through a 0.45-µm filter (ADVANTEC MFS, Inc.,

141

Tokyo, Japan), and used for the quantitative analysis of chlologenic acid, flavonols, and DNJ.

142

The mineral element quantities in mulberry leaves were measured. The total nitrogen was

143

measured by the Kjeldahl procedure22. Other mineral elements were extracted with 1 mol/L

144

hydrochloric acid after dry ashing, including phosphorus by the vanado-molybdate method,

145

potassium by flame photometry, and calcium and magnesium by the atomic absorption

146

method, according to a previously published method 22.

147 148

Quantification of chlorogenic acid, flavonols, and DNJ

149

Chlorogenic acid, flavonols, and DNJ in mulberry leaves were analyzed using a high

150

performance liquid chromatograph (Waters HPLC system, Waters Corporation, Milford, MA,

151

USA) equipped with an Alliance separation module 2695, a photodiode array detector 2996,

152

an F-3010 fluorescence spectrophotometer (Hitachi Instruments, Inc., Tokyo, Japan), and an

153

ODS Wakosil-II 5C18 RS column (4.6 × 250 mm; Wako Chemicals, Inc., Osaka, Japan).

154

To analyze the chlorogenic acid and the flavonol contents, the column temperature was

155

set at 40°C, and a 20% acetonitrile solvent containing 0.1% formic acid at a flow rate of 1

156

mL/min was used. The wavelengths detecting chlorogenic acid and flavonols were 280 nm

157

and 350 nm, respectively. DNJ was assessed using the method of Kim et al 23. The standard

158

flavonols, kaempferol 3-(6-rhamnosylglucoside), quercetin 3-(6-malonylglucoside), and

159

kaempferol 3-(6-malonylglucoside), were purified from the mulberry leaves according to the

160

method of Katsube et al. 7. Rutin was purchased from Wako Chemicals Inc. (Osaka, Japan),

161

and isoquercitrin, astragalin and DNJ were purchased from Funakoshi, Inc. (Tokyo, Japan).

162

The total flavonol content in the mulberry leaves of ‘Ichinose’ was expressed as the sum of six


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flavonol glycosides: rutin, isoquercitrin, kaempferol 3-(6-rhamnosylglucoside), quercetin

164

3-(6-malonylglucoside), astragalin, and kaempferol 3-(6-malonylglucoside).

165 166

Statistical analysis

167

Statistical analysis of data was performed using version 9.0 JMP statistical analysis

168

software (SAS Institute, Tokyo, Japan). Results were expressed as the means ± standard error

169

(SE). Data were tested by a one-way analysis of variance, followed by Tukey–Kramer’s test

170

for multiple comparisons.

171 172

Results and Discussion

173

Field study

174

Table 2 shows the chemical properties of soil in each common cultured field. The

175

carbon/nitrogen ratio of Field A (ranging from 14 to 17) was higher than that in other fields

176

(ranging from 11 to 17). The levels of total-carbon and total-nitrogen in Fields B and C were

177

lower than those in Fields A and D. Based on these facts, and the low cation exchange

178

capacity and cation exchangers of Fields B and C, the fertility of Field B was considered

179

inferior to those of the other fields. The difference in fertility between Fields A and B,

180

although both were located on the Gonokawa River, was of interest because Field A was

181

sometimes covered with humus-containing soil, due to flooding after heavy rains, while Field

182

B was not, due to the presence of the river bank.

183

Table 3 shows the mineral elements in mulberry leaves at the four common cultured

184

fields. The nitrogen content of Field A was markedly higher than those of the other fields.

185

Generally, the average nitrogen content in mulberry leaves is ~4%; however, only Field A

186

showed a sufficient nitrogen content. The low nitrogen contents of Fields B and C at 3.31%



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and 3.26%, respectively, were probably to the result of lower nitrogen contents in the

188

mulberry trees.

189

Table 4 shows functional components in mulberry leaves at four common cultured fields.

190

The chlorogenic acid content in Field C was significantly higher than those in Fields A and D,

191

but the flavonol content in Field C was significantly lower than those in Fields A and B. The

192

DNJ content was highest in Field A, and those of Fields B and C were very low. Among the

193

examined functional components, the DNJ content in mulberry leaves varied greatly among

194

the cultured fields. The ratios of the highest to lowest chlorogenic acid values and the flavonol

195

content were approximately 1.2-fold, and that of the DNJ content was 1.6-fold. The functional

196

component contents in mulberry leaves vary widely owing to varietal and annual differences,

197

as well as harvest times

198

differences in the functional component contents among the fields were relatively small.

199

Therefore, the effects of soil texture and soil chemical properties on the functional

200

components were considered to be small in the common cultured fields.

3,11,13,15-17

, and these difference can be several fold. In our study, the

201

Table 5 shows the correlation coefficients between the functional components and the

202

mineral elements in all of the mulberry leaves sampled from the four common cultured fields.

203

The highest correlation was noted between the DNJ and nitrogen contents (0.60). Additionally,

204

the nitrogen content showed a strong negative correlation with chlorogenic acid, although it

205

showed a weak correlation with the flavonol content (0.14). Moreover, the calcium content

206

was negatively correlated with the chlorogenic acid content (−0.44), but was positively

207

correlated with the DNJ content (0.47). Among the examined mineral elements in mulberry

208

leaves, the effects of the nitrogen content were found to be the greatest. The nitrogen content

209

in mulberry leaves can be influenced by the amount of nitrogen applied and the soil fertility.

210

Therefore, the differences in the chlorogenic acid and DNJ contents among the four cultured


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fields in our study were considered to be attributed to soil fertility. Based on our results, it was

212

suggested that the amounts of applied nitrogen and calcium could affect the functional

213

components in mulberry leaves.

214 215

Effects of applied nitrogen amounts on the functional components of mulberry leaves

216

Based on our common cultured field study results on the relationship between nitrogen

217

levels and the functional components in mulberry leaves, we further investigated the effects of

218

applied nitrogen amounts on specific functional components using mulberry trees cultured in

219

plastic pots and in experimental fields.

220

Figure 1 shows nitrogen levels and functional components in mulberry leaves relative to

221

the applied nitrogen amounts. Five experimental groups were defined by the amounts of

222

applied nitrogen. The nitrogen content of mulberry leaves significantly increased in

223

accordance with the applied nitrogen amount. Conversely, the chlorogenic acid content

224

decreased in accordance with the applied nitrogen amount. The chlorogenic acid content of

225

Group I was 2.5-fold that of Group V. The flavonol content showed a similar trend to that of

226

the chlorogenic acid content, although the difference was relatively small at ~1.6-fold. In

227

contrast, the DNJ content increased in accordance with the applied nitrogen amount. The

228

difference in the DNJ contents between Groups I and V was 2.8-fold, which was the greatest

229

among the examined four functional components. The functional components showed the

230

greatest differences between Groups I and II, when the applied nitrogen amounts were small.

231

Although our pot cultivation showed that the flavonol content decreased as the

232

nitrogen application amount increased, such a negative correlation was not shown in the

233

common cultured field (Table 5). The difference between the pot experiment and field study

234

results could be attributed to the nitrogen content in mulberry leaves. A wide range of nitrogen



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contents in leaves was seen in the pot experiment (1.44 to 4.19%) but the range was small in

236

the field study (3.26 to 4.14%). As in the pot experiment, when the applied nitrogen amount

237

was small, there was a greater difference in the mulberry leaves’ flavonol content than when

238

the applied nitrogen amount was large. In contrast, in the field culture, the nitrogen

239

application had much less impact, especially when the applied nitrogen amount was large.

240

To effectively increase the functional component amounts in the entire cultivation field,

241

we first needed to increase the mulberry leaf yield per tree. Therefore, we investigated the

242

functional component levels per mulberry tree using pot-cultured samples (Table 6). The fresh

243

leaf weights per mulberry tree in Groups I (lowest applied nitrogen amount) and V (highest

244

applied nitrogen amount) were markedly less than those of Groups II and III. The cause of the

245

leaf yield reduction in Group I was the growth inhibition caused by the nitrogen deficiency,

246

with the leaf nitrogen content being only 1.44%. Whereas, the leaf yield reduction of Group V

247

was caused by the excessive nitrogen application, leading to a loss of leaves and the partial

248

discoloration (browning) of leaves. The total amount of the functional components per tree

249

was calculated using fresh leaf weights, functional component contents, and water contents in

250

leaves (I: 74%, II: 75%, and III–V: 76%). Group I had the highest chlorogenic acid content,

251

Group II had the highest flavonol content, and Group III had the highest DNJ content. Since

252

the chlorogenic acid and flavonol contents in leaves increased with reduced nitrogen

253

application levels, leading to reduced leaf yields, the applied nitrogen amount needs to be

254

adjusted to balance between the leaf yield and functional component contents. The difference

255

between the highest and lowest DNJ content in leaves was 2.8-fold (Figure 1), and that of the

256

DNJ content per tree was 4.2-fold (Table 6). This was considered because the applied nitrogen

257

amount was suitable not just for growth and leaf yield, but for the DNJ content per tree.

258

Therefore, the optimal nitrogen fertilization amount for leaf yield is considered to be optimal


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for the DNJ content.

260

Table 7 shows the effects of the applied nitrogen amount on the appearance and yields

261

of mulberry trees in the experimental field. Four groups were defined by the levels of their

262

nitrogen applications. Branch length and branch yield showed almost the same results,

263

irrespective of the applied nitrogen amount. Leaf colors in the 2011 summer yield and in the

264

2012 autumn yield were similar in all of the groups, irrespective of the applied nitrogen

265

amount, but those in the autumn of 2011 and in the summer of 2012 were darker in

266

accordance with the nitrogen application amount, and this tendency was marked in the 2011

267

autumn yield.

268

Figure 2 shows the effects of the applied nitrogen amount on the functional

269

components of mulberry leaves in the experimental field. In the 2011 summer yield, the

270

functional component levels did not show significant differences among the four groups,

271

except for the DNJ content of Group III. In the latter cropping season (from autumn 2011 to

272

2012), the nitrogen content in mulberry leaves increased in accordance with the applied

273

nitrogen amount. Conversely, the chlorogenic acid and flavonol contents decreased as the

274

applied nitrogen application increased. The DNJ content was not different among the four

275

groups until the 2012 summer yield, and it was notably higher in Groups II and III compared

276

with Groups O and I in the autumn of 2012. The difference between the highest and lowest

277

values was the greatest for the nitrogen (0.58%) and DNJ [37 mg/100 g dry weight (DW)]

278

contents in the autumn of 2012, and for the chlorogenic acid (217 mg/100 g DW) and flavonol

279

(239 mg/100 g DW) contents in the autumn of 2011. The annual and cropping season

280

differences were greater than the applied nitrogen differences.

281

The relationship between the nitrogen application level and the functional components

282

in the experimental field showed the same trend as in the pot culture. The contents of



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chlorogenic acid, flavonol, and DNJ differed depending on the year and cropping season.

284

Nakanishi et al. 17 reported that the DNJ content in mulberry leaves was different depending

285

on the sampling time, and our previous study showed that solar radiation influences flavonol

286

and DNJ components in mulberry leaves 12. Thus, the annual and seasonal differences in the

287

functional component levels in the present study were considered to be influenced by the

288

cultivation season and climatic environment. However, the relationship between the applied

289

nitrogen amount and each functional component tended to be the same in each treatment

290

group; therefore, the impact of applied nitrogen levels on the resulting functional components

291

is believed to be great. Interestingly, in the autumn yield of 2011, which had darker leaf colors,

292

the greatest differences between the highest and lowest chlorogenic acid and flavonol contents

293

were noted by the applied nitrogen amounts. The leaf color was directly affected by the

294

applied nitrogen amount. Thus, in the future, mulberry leaf colors may be evaluated as

295

indicators for chlorogenic acid and flavonol contents.

296

Mudau et al.

19

reported that the polyphenolic content of bush tea increased after

297

nitrogen application, while Stewart et al. 20 reported that the flavonol levels in plant tissues

298

increased with lower nitrogen applications. Our results showed the same trend as those of

299

Stewart et al. Antioxidant components, including flavonol, are induced by various stresses,

300

and our previous study showed that the flavonol content increases after mulberry exposure to

301

UV light stress 12. Our present study also suggests that a nitrogen deficiency stress induced an

302

increase in the chlorogenic acid and flavonol contents in mulberry leaves. Kimura et al. 3 and

303

Nakanishi et al. 16 reported that the DNJ content increases in the summer and in the upper

304

branches of mulberry trees. Shading from solar radiation increases DNJ levels in mulberry

305

trees; thus, we hypothesized that DNJ is a metabolic product induced by stress alleviation 12.

306

In addition, DNJ increases to protect mulberry leaves from insect herbivores. Konno 24 and


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Nakanishi et al. 16 reported that the DNJ content increased in some portions of mulberry

308

branches during a remarkable growth period to protect themselves from insects. Based on our

309

present study on nitrogen applications, we believe that flavonol production is induced by the

310

stress of insufficient nitrogen levels and that DNJ production is induced by less stress when

311

sufficient nitrogen is applied.

312

Our study showed that the chlorogenic acid, flavonol, and DNJ contents were affected

313

by the amount of applied nitrogen. Namely, a lower applied nitrogen level leads to increased

314

chlorogenic acid and flavonol contents, and a higher nitrogen application level leads to an

315

increased DNJ contents. We believe that selectively increasing individual functional

316

components in mulberry leaves is possible, by adjusting the optimal level of nitrogen

317

fertilization.

318 319

Acknowledgments

320

The authors would like to thank Yukikazu Yamasaki, Shimane University, Japan, for

321

his valuable guidance in preparing our manuscript. We also appreciate Junko Fujimoto,

322

Shimane Agricultural Technology Center, Japan, for her assistance in the preparation of the

323

fertilizer experiment.

324 325

Funding sources

326

We certify that no funds were provided for any of the authors in relation to this study from

327

outside sources.

328



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of arachidonic acid pathway by mulberry leaf extract. Inflammopharmacology 2015, 23,

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Page 20 of 30

20: Figure captions Figure 1. Nitrogen and functional component levels in mulberry leaves relative to the amount of nitrogen applied to potted plants. A: Nitrogen, B: Chlorogenic acid, C: Flavonol, and D: DNJ. Amounts of applied ammonium sulfate per culturing pot on April 14th, June 9th, and July 2nd: Group I = 1 g, Group IV = 2.5 g, Group III = 5 g, Group IV = 10 g, and Group V = 15 g. On July 26th, completely opened leaflets of the youngest opened leaves, and the subsequent two leaves on the same branch, were sampled. Data are expressed as means ± SE (n = 8). The same lowercase letters indicate no significant difference (P < 0.05).

Figure 2. Effects of applied nitrogen amounts on the nitrogen content and functional components of mulberry leaves in experimental fields. A: Nitrogen content, B: Chlorogenic acid, C: Flavonol, and D: DNJ Four experimental groups were formed based on the amounts of applied ammonium sulfate, which provides nitrogen: O: 0 kg, I: 6 kg, II: 15 kg, and III: 30 kg per 1,000 m2. The youngest opened leaves with completely opened leaflets, and the subsequent two leaves of two branches with moderate growth, were sampled from each stock. Data are expressed as means ± SE (n = 20). Significant differences are indicated by different lowercase letters (P < 0.05) in the same harvesting season.


Page 21 of 30


21: Tables

Table 1. Fertilization conditions of the four common cultured fields

Applied fertilizer (kg/a)

Timing of fertilization

N

P2O5

K2O

Type of fertilizer

Composta

Field A

December-March

-

-

-

-

200

Field B

December-March

-

-

-

-

200

March

1.00

0.40

0.40

Chemical fertilizer

June

0.60

0.24

0.24

Chemical fertilizer

-

Total

1.00

0.40

0.40

April

1.05

0.49

0.56

Slow release fertilizer

-

Field C

Field D

a

Swine manure was used as compost (containing N: 1.29%; P2O5: 1.64%; K2O: 1.3%).



Page 22 of 30

22: Table 2. Chemical properties in each soil layer of the four common cultured fields

Field A

Field B

Field C

Field D

CaO

MgO

K2O

Available phosphoric acid (mg/100g)

19.5

265

51

52

16

14

15.2

209

44

15

26

0.02

17

5.4

67

18

15

9

0.80

0.07

11

7.3

89

33

29

48

0.05

0.40

0.03

13

6.0

80

24

14

17

5.6

0.05

1.32

0.11

12

11.1

128

20

17

41

14-33

4.1

0.21

0.70

0.06

12

9.7

49

9

26

45

3

33-

3.8

0.22

0.46

0.04

12

8.1

18

2

28

19

1

0-14

7.0

0.21

2.93

0.28

11

16.4

563

33

75

77

2

14-33

7.0

0.07

0.63

0.06

11

10.5

195

19

53

76

Soila layer

Depth (cm)

pH (H2O)

ECb (mS/cm)

Total carbon (%)

Total nitrogen (%)

C/Nc ratio (%)

CECd (me/100 g)

1

0-6

5.9

0.08

4.55

0.30

15

2

6-24

6.0

0.06

2.64

0.19

3

24-48

6.4

0.02

0.34

1

0-18

6.1

0.10

2

18-32

6.4

1

0-14

2

a

Layer 1 = upper layer; layer 2 = middle layer; layer 3 = lower layer

b

EC = electro-conductivity

c

Cation exchanger (mg/100 g)

C/N = carbon/nitrogen ratio, which was calculated by dividing the total carbon by the total nitrogen applications

d

CEC = cation exchange capacity


Page 23 of 30


23: Table 3. Mineral elements in mulberry leaves from the four common cultured fieldsa N (%)

P (%)

K (%)

Ca (%)

Mg (%) 0.23 ± 0.04 b

Field A

4.14 ± 0.28 a

0.41 ± 0.05 b

2.95 ± 0.14 a

2.02 ± 0.24 a

Field B

3.31 ± 0.24 b

0.56 ± 0.10 a

2.89 ± 0.17 ab

1.82 ± 0.32 ab 0.29 ± 0.03 a

Field C

3.26 ± 0.30 b

0.45 ± 0.06 b

2.54 ± 0.21 c

1.18 ± 0.19 c

0.22 ± 0.03 b

Field D

3.40 ± 0.36 b

0.42 ± 0.08 b

2.66 ± 0.36 bc

1.78 ± 0.32 b

0.20 ± 0.02 b

a

Two leaves, which were located at the one third position from the branch top, were sampled

from one branch per stock of normal growth. Data are expressed as means ± SE (n = 20 in Fields A and D, n = 10 in Fields B and C). The same lowercase letters indicate no significant difference (P < 0.05).



Page 24 of 30

24: Table 4. Functional components in mulberry leaves from the four common cultured fieldsa Chlorogenic acid (mg/100 gDW)

a

Flavonol (mg/100 gDW)

DNJ (mg/100 gDW)

Field A

879 ± 29

b

1374 ± 40

a

180 ± 6

a

Field B

995 ± 42

ab

1318 ± 59

a

128 ± 5

cd

Field C

1070 ± 66

a

1122 ± 48

b

111 ±7

d

Field D

897 ± 36

b

1287 ± 34

ab

148 ± 4

bc

Two leaves, which were located at the one third position from the branch top, were sampled

from one branch per stock of normal growth. Data are expressed as means ± SE (n = 20 in Fields A and D, n = 10 in Fields B and C). The same lowercase letters indicate no significant difference (P < 0.05).


Page 25 of 30


25: Table 5. Correlation coefficients between the functional components and mineral elements in mulberry leavesa

N Chlorogenic acid

b

-0.48 **

P

K

Ca

Mg

0.22

-0.07

-0.44 **

0.17

Flavonol

0.14

0.08

0.26 *

0.24

0.03

DNJ

0.60 **

-0.34 *

0.19

0.47 **

-0.21

a

b

Leaves (n = 60) were sampled from four common cultured fields.

Significant differences are shown (** P < 0.01, * P < 0.05).



Page 26 of 30

26: Table 6. The functional component amounts per mulberry tree relative to the applied nitrogen levelsa

a

Functional componentsb (mg/stock)

Nitrogen treatment

Fresh leaf yield (g/stock)

Chlorogenic acid

Flavonol

DNJ

I

170 ± 10 b

503

764

25

II

287 ± 18 a

479

949

83

III

319 ± 17 a

397

934

106

IV

285 ± 13 a

310

777

101

V

188 ± 15 b

193

469

73

Amount of applied ammonium sulfate per culturing pot on April 14th, June 9th, and July

2nd : Group I = 1 g, Group IV = 2.5 g, Group III = 5 g, Group IV = 10 g, and Group V = 15 g. On July 26th, completely opened leaflets of the youngest opened leaves, and the subsequent two leaves on the same branch, were sampled. Data are expressed as means ± SE (n = 8). The same lowercase letters indicate no significant difference (P < 0.05). b

The functional component amounts per tree were calculated based on fresh leaf weight,

functional component amount, and water content in leaves (I: 74%, II: 75%, and III–V: 76%).


Page 27 of 30


27: Table 7. Effects of applied nitrogen amounts on the appearance and yields of mulberry treesa

Harvesting Nitrogen season treatment

Summer 2011 Autumn

Summer 2012 Autumn

a

Longest branch (cm)

Yields (g/stock)

Leaf colorb

O

186 ± 2

a

207 ± 2

a

38.0 ± 0.5

a

I

186 ± 2

a

1688 ± 73

a

38.0 ± 0.4

a

II

189 ± 3

a

1724 ± 89

a

37.8 ± 0.7

a

III

185 ± 3

a

1637 ± 76

a

37.8 ± 0.5

a

O

186 ± 4

a

1785 ± 110 a

36.6 ± 0.4

c

I

190 ± 5

a

1884 ± 113 a

37.4 ± 0.5

bc

II

189 ± 4

a

1856 ± 103 a

38.9 ± 0.4

ab

III

188 ± 3

a

1884 ± 111 a

39.3 ± 0.3

a

O

182 ± 4

a

1900 ± 83

a

31.8 ± 0.5

b

I

181 ± 3

a

1862 ± 101 a

33.1 ± 0.4

ab

II

188 ± 3

a

1966 ± 108 a

33.8 ± 0.3

a

III

184 ± 3

a

2000 ± 120 a

34.5 ± 0.4

a

O

193 ± 4

b

1468 ± 67

a

36.4 ± 0.5

a

I

201 ± 3

ab

1517 ± 91

a

37.5 ± 0.4

a

II

209 ± 2

a

1574 ± 79

a

36.7 ± 0.4

a

III

207 ± 2

a

1522 ± 71

a

37.7 ± 0.3

a

Four experimental groups were formed based on the amount of applied ammonium sulfate,

which provides nitrogen: O: 0 kg, I: 6 kg, II: 15 kg, and III: 30 kg per 1000 m2. The branches were harvested on July 11th and October 4th in 2011, and on July 9th and September 19th in 2012. Data are expressed as means ± SE (n = 20). The same lowercase letters indicate no significant difference (P < 0.05) during the same harvesting season. bLeaf color was evaluated using a SPAD 502 chlorophyll meter.



Page 28 of 30

28: 5.0 4.0

a b

3.0

b

c

2.0

B Chlorogenic acid contents (mg/100gDW)

A Nitrogen contents (%)

Figure graphics

d

1.0 0.0

Flavonol contents (mg/100gDW)

C

2000

II

III

IV

900 ab 600

bc

c

c

III

IV

V

ab

a

IV

V

300 0 I

II

D

a

160 b

1500

a

V

bc

cd

d

1000 500

DNJ contents (mg/100gDW)

I

1200

b c

120 80

d

40 0

0 I

II

III

IV

V

I

Figure 1.


II

III

Page 29 of 30


29: a

a

a

b

a

a

b

a

ab

b

d

a

c

2.0 1.0 0.0 O I

II III O I

Summer

II III O I

Autumn

II III O I

Summer

2011

C 1800

a

a

a a a

Flavonol contents (mg/100gDW)

1600

a

ab

b

1000

b

c

bc

800 600 400 200 0 II III O I

II III O I

Autumn

II III O I

Summer 2012

a b

b

II III

Autumn

b

a

a b b

400 200 0 O I

II III O I

II III O I

Autumn

II III O I

Summer

2011

c

a

c

600

Autumn

b b

bc

800

D 300 bc

ab

a

Summer

a

2011

a

2012

1200

Summer

a

II III

1400

O I

a

1200 1000

c

3.0

B 1400 Chlorogenic acid contents (mg/100gDW)

a

a

DNJ contents (mg/100gDW)

Nitrogen contents (%)

A 4.0

a

a

II III

Autumn

2012

ab a

b

250

b

a

b

a

200 a

a

a

a

a

a a

150 100 50 0 O

I

II III O I

Summer 2011

Figure 2.


II III O I

Autumn

II III O

Summer 2012

I

II III

Autumn


Page 30 of 30

30: Graphic for table of contents


Effects of Applied Nitrogen Amounts on the Functional Components of

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