Herbicidal Spectrum, Absorption and Transportation, and

Subscriber access provided by UNIV OF TEXAS ARLINGTON

Article

Herbicidal spectrum, absorption and transportation, physiological effect to Bidens pilosa of natural alkaloid, berberine Jiao Wu, Jing-jing Ma, Bo Liu, Lun Huang, Xiao-qing Sang, and Li-juan Zhou J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b01259 • Publication Date (Web): 12 Jul 2017 Downloaded from http://pubs.acs.org on July 12, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 51

Journal of Agricultural and Food Chemistry

1 2

Herbicidal spectrum, absorption and transportation, physiological effect to Bidens pilosa of natural alkaloid, berberine

3 4

Jiao Wu,|| Jing-Jing Ma,|| Bo Liu, Lun Huang, Xiao-Qing Sang, and Li-Juan Zhou*

5

Key Lab of Natural Pesticides & Chemical Biology, Ministry of Education,

6

Department of Pesticide Science, South China Agricultural University, Guangzhou,

7

Guangdong, China, 510642

8 9 10

* Correspondence to:

11

Li-Juan Zhou

12

Key Lab. of Natural Pesticides & Chemical Biology, Ministry of Education

13

South China Agricultural University, Guangzhou, Guangdong, China,

14

Phone: 86 15917363981

15

E-mail: [email protected]

16 17 18 19 20 21 22

1

ACS Paragon Plus Environment


23

ABSTRACT

24

Berberine is a natural herbicidal alkaloid from Coptis chinensis Franch. Here we

25

characterized its herbicidal spectrum, absorption and transportation in plant, and the

26

possible mechanism. Berberine showed no effect on the germination of the 10 tested

27

plants. The IC50 values of berberine on the primary root length and fresh weight of the

28

10 tested plants ranged from 2.91 to 9.79 mg L−1 and 5.76 to 35.07 mg L−1,

29

respectively. Berberine showed a similar herbicidal effect to Bidens pilosa as the

30

commercial naturally derived herbicide cinmethylin. HPLC and fluorescence analysis

31

revealed that berberine was mainly absorbed by B. pilosa root and transported through

32

vascular bundle acropetally. Enzyme activity studies, GC-MS analysis, SEM and

33

TEM observations indicated that berberine might firstly function on cell membrane

34

indicated by variation of the IUFA percent and then cause POD, PPO, and SOD

35

activity changes and cellular structure deformity, which was eventually expressed as

36

the decrease of cell adaptation ability and abnormal cell function and may even result

37

in cell death. Environmental safety evaluation tests revealed that berberine was low in

38

toxicity to Brachydanio rerio. These indicate that berberine has potential to be a

39

bioherbicide and/or a lead molecule for new herbicides.

40 41

KEYWORDS: berberine, Bidens pilosa L., herbicidal spectrum, absorption,

42

transportation, physiological effect, mechanism 2


Page 2 of 51

Page 3 of 51


43

INTRODUCTION

44

Weeds are plants whose undesirable qualities outweigh their good points, at

45

least according to humans. Weeds compete with crops for space, nutrients, water and

46

light, and therefore cause substantial economic loss. Traditionally, synthetic

47

herbicides are widely used and have become a major control method for weed

48

management. Herbicides provide a convenient, economical, and effective way to

49

help manage weeds. However, the continuous use of traditional synthetic herbicides

50

leads to various negative impacts such as weed resistance to herbicides.1 Therefore,

51

there is an urgent need for the development of alternatives to traditional synthetic

52

herbicides which are much safer and with new mode of actions. Generally, natural

53

products are particularly attractive as templates because they occupy a wider

54

chemical space with greater structural diversity than traditional synthetic

55

compounds.2

56

Bioherbicides are biologically-based control agent for weeds. They can be used

57

directly and have been used as a model for the development of several successful

58

herbicides (glufosinate and the triketone herbicides) that introduced new mechanisms

59

of action, which are greatly needed to overcome the acquired resistance to synthetic

60

herbicides in agricultural production. There is, therefore, a great interest in exploring

61

natural products to develop new bioherbicides.3 Herbicidal effects of many natural

62

chemicals have been studied. These chemicals include quinones (e.g., strigol,

63

sorgoleone, juglone), flavonoids (e.g., catechin, ceratiolin, chalcones, kulkulkanin B

64

and heliannones), phenols (e.g., heliannuols, ellagic acid, usnic acid), ethers (e.g.,

3



65

cyperin, heliespirone A, helianane), terpenoids (e.g., 1,4-cineole, 1,8-cineole, geranial,

66

citral, solstitiolide, messagenic acids, melilotigenins, breviones, germacranolides,

67

guaianolides, parthenolide, costunolide, ailanthone, artemisinin, trichocaranes,

68

chaparrine, ailanthinol B), coumarins (e.g., coumarin, psoralen), benzoxazinoids (e.g.,

69

hydroxamic acids), glucoinolates (e.g., glucohirsutin, hirsutin, arabin), fatty acids (e.g.,

70

pelargonic acid), amides (e.g., sarmentine), alkaloids (e.g., fagomine), some amino

71

acids such as meta-tyrosine and some essential oils such as clove oil and lemongrass

72

oil.4-6

73

Included in these chemicals are alkaloids, which are naturally-occurring

74

nitrogen-containing biologically active heterocyclic compounds. Over the last few

75

years, a large number of biologically important alkaloids with antiviral, antibacterial,

76

anti-inflammatory, antimalarial, antioxidant and anticancer activities have been

77

isolated from various biosources.7 However, there are few assessments of the

78

herbicidal activities of natural alkaloids.8

79

Berberine (chemical formula: C20H18NO4. Refer to Fig. 1), a benzylisoquinoline

80

alkaloid, is a major component present in numerous medicinal plants which are used

81

for treating diarrhea and other gastrointestinal diseases in Asian countries. Berberine

82

is known to have a wide range of biological activities such as antioxidant,

83

antibacterial, antifungal, antiviral, anti-inflammatory, anti-tumor, anti-diarrhoeal,

84

anti-dyslipidemic and anti-diabetic properties.9 In addition to its pharmaceutical

85

activities, berberine is also used in agriculture as insecticide and fungicide.10-11

86

Interestingly, it was also found to possess herbicidal activity as a result of a

4


Page 4 of 51

Page 5 of 51


87

bioactivity-guided isolation of the herbicidal active components from the rhizomes

88

of Coptis chinensis Franch.12 We previously reported that extracts of C. chinensis

89

exhibited significant inhibition of root growth in some weeds through the active

90

component berberine.13 In this study, we further explored the herbicidal activities of

91

berbine on monocot and dicot weeds and characterized the mode of inhibition by

92

examining its absorption site, transport pattern and the physiological effects and

93

evaluating its ecological safety. Here we show that berberine possessed non-selective

94

herbicidal activity and mainly was absorbed by roots and transported from stem

95

vascular bundle acropetally to leaves and showed low toxicity to Brachydanio rerio.

96 97

MATERIALS AND METHODS

98

Herbicidal activities of berberine on some monocot and dicot plants.

99

The herbicidal effects of berberine (Sigma, CAS:141433-60-5) were determined

100

using dicot and monocot plants. The seeds of weeds were collected on the campus of

101

South China Agricultural University. These species are important components of the

102

herbaceous vegetation in crop fields and forests, especially, Eucalyptus forests, of

103

southern provinces in China.

104

Fungal and bacterial control was accomplished by treating the plant seeds (5 dicots:

105

Ageratum conyzoides, Mikania micrantha, Celosia argentea, Amaranthus retroflexus and

106

Bidens pilosa; 5 monocots: Echinochloa crusgalli, Dactyloctenium willd, Eleusine indica,

107

Triticum aestivum and Oryza sativa) with 1.0% (v/v) sodium hypochlorite for 10 min.

108

Then the seeds were washed 2-3 times by the tapping water. Berberine was applied in

5



109

distilled water at concentrations from 2 to 32 mg L−1 to glass beakers containing 20 seeds

110

of a plant species underlain by Whatman #2 filter paper. Berberine solutions (8 mL) were

111

introduced into the glass beakers. Then, the glass beakers, sealed with polyethylene

112

wrapping film with several little holes for ventilation, were placed in a growth chamber

113

calibrated to provide 12 h light/12 h darkness at 25±2 °C. Distilled water only was

114

introduced as a control. A completely randomized design was selected and all treatments

115

were replicated at least three times. The germination rate was calculated each day after

116

the treatment. The primary root length and fresh weight were measured at 7 d after the

117

treatment. Root inhibition rate was calculated by the following formula: root inhibition

118

rate (%) = ((root length of control-root length of treatment) / root length of control ) ×

119

100.14

120

The herbicidal effects of berberine at 10 mg L−1 on duckweed (Lemna minor)

121

were determined by the method described by Iwasa (2000).15 Hogland nutrient

122

solution was used as a nutrient-supplemented liquid medium. Berberine was applied

123

to the plastic bowls containing 30 L. minor plants in Hoagland’s media at final

124

concentrations 10 mg L−1 and placed in an environmental growth chamber calibrated

125

to provide 12 h light/12 h darkness at 25±2 °C. The fresh weight of each replicate was

126

investigated before the treatment. Eighteen replicates in each treatment. Three

127

randomly selected replicates in each treatment were used to evaluate the fresh weight,

128

thallus number, contents of chlorophyll a, chlorophyll b and total chlorophyll 3, 5, 7,

129

and 9 d after the treatment. Visual results were presented in a 6 -well cell culture plate.

130

Glyphosate, cinmethylin, sulcotrione and 2,4-D were used as contrast herbicides

6


Page 6 of 51

Page 7 of 51


131

to show the herbicidal effect of the natural product berberine. Achenes of B. pilosa

132

were pretreated as above. Berberine and glyphosate (Dikma Technologies, CAS:

133

1071-83-6, 95.5%) were dissolved in water while 2,4-D (Sigma, CAS: 94-75-7, 98%)

134

were dissolved in ethanol to make 5000 mg L−1 stock solution and then diluted with

135

water to get series of concentrations from 2 to 32 mg L−1. Cinmethylin (Sigma, CAS:

136

87818-31-3, 98.7%) and sulcotrione (Sigma, CAS: 99105-77-8, 98.8%) were

137

dissolved in acetone to make 2000 mg L−1 stock solution and then diluted with water

138

to get series of concentrations from 2 to 32 mg L−1. A similar set of experiments were

139

conducted for the seeds treated with sterile water only for berberine or glyphosate, or

140

treated with ethanol (0.64%) for 2,4-D to serve as controls. The primary root length

141

and fresh weight were investigated at 14 d after incubation.16 All experiments were

142

repeated at least three times.

143

Absorption and transport of berberine in B. pilosa seedling

144

Main absorption site was determined by applying berberine on B. pilosa

145

seedlings (4-leaf stage) at a final concentration of 32 mg L−1 by root incubation,

146

stem-coating and leaf-dipping. Primary root length, plant height, whole plant fresh

147

weight and root fresh weight were measured at 1, 2, 3, 5 and 7 d after incubation.

148

Transport pattern of berberine in B. pilosa was determined by the method

149

described previously by Kamal et al.17 Specifically, B. pilosa seedlings (4-6-leaf stage)

150

were treated by root incubation with 32 mg L−1 berberine or by leaf-painting with

151

1000 mg L−1 berberine (40 µL on four new leaves of each B. pilosa, nine plants in

152

each pot). Then the content of berberine in stem, leaf, root and the incubation solution

7



153

was detected by HPLC (Agilent 1100, Santa Clara, CA) at different time intervals (0.5,

154

1, 2, 3, 5 and 7 d after the treatment). Chromatographic analysis was investigated on a

155

Spherisorb 5HC-C18, 250 mm×4.6 mm (Agilent) column. Detection was performed

156

with a UV detector. Acetonitrile + 0.05mol L-1 sodium dihydrogen phosphate (25 + 75

157

by volume) was used as the mobile phase. The injection volume of sample was 10.00

158

µL and the flow rate was kept at 1 mL min-1. Column temperature was

159

30 °C.Berberine has an excitation wavelength of 405 nm and emission at 520 nm.18

160

Transport of berberine was further confirmed in B. pilosa by fluorescence microscope.

161

Visualization of uptake and transportation of berberine was performed on 20 d old B.

162

pilosa seedlings. Specifically, roots, stems and leafs were collected at 0.5, 1, 2, 3 and

163

5 d after root incubation treatment with 32 mg L−1 berberine and washed thoroughly

164

with water to remove the residue. Free-hand cross-sections of root, stem and petiole

165

were prepared.19 Then sections were examined using fluorescence microscopy (Nikon

166

DS-Ri1). The presence of fluorescence at the transportation site is positive for

167

berberine. All experiments were repeated at least three times.

168

Analysis of B. pilosa root treated with berberine.

169

The sterilized seeds were planted in 10×10 cm Petri dish containing Hoagland's

170

media. Berberine was added to the Hoagland's media at final concentrations ranging

171

from 2 to 32 mg L−1. Growth conditions were described as above.

172

Fresh root samples (0.2 g) were ground in 1 mL phosphate buffer solution (0.1

173

mol L-1, pH7.8) in a mortar precooled to -20 °C, and then centrifuged at 12000 rpm for

174

15 min at 4 °C. The supernatant was used to detect the relative factors.

8


Page 8 of 51

Page 9 of 51


175

The root vigor of B. pilosa root was measured using triphenyl tetrazolium

176

chloride (TTC) as described by Ruf et al.20 The activities of superoxide dismutase

177

(SOD), polyphenol oxidase (PPO) and peroxidase (POD) were measured according to

178

the methods by Zhang et al.,21 Thipyapong et al.,22 and Zipor et al.23

179

Oxidative damage to root lipids, resulting from berberine, was estimated by the

180

content of total 2-thiobarbituric acid reactive substances (TBARS) expressed as

181

equivalents of malondialdehyde (MDA). The TBARS content was estimated by the

182

method of Hajlaoui et al.24 with some modifications. Fresh root samples (0.5 g) were

183

ground in 5 mL of 10% (w/v) trichloroacetic acid (TCA). Following the centrifugation

184

at 4000 rpm for 10 min, an aliquot of 1 mL from the supernatant was added to 1 mL

185

of 0.6% (w/v) thiobarbituric acid (TBA). The samples were heated at 100 °C for 15

186

min. Thereafter, the reaction was stopped by cooling down in an ice water bath. After

187

centrifugation, the absorbance of the supernatant was read at 532 nm on a

188

spectrophotometer (Model Camspec M330 UV/Vis) and corrected for non-specific

189

turbidity by subtracting the absorbance at 600 nm. The concentration of

190

malondialdehyde was calculated using the extinction coefficient of 155 mm cm-1.

191

Fatty acid (FA) composition was determined by gas chromatography-mass

192

spectrometry (GC-MS). Total fatty acids were converted into their methyl esters using

193

sodium methylate, according to the method described by Bettaieb et al.25 Individual

194

FA were separated and quantified by GC-MS using a model Agilent Technologies

195

6890N network GC system equipped with an Agilent Technologies 5975 inert XL

196

mass selective detector. The test columns all had nominal dimensions of 30 m × 250

9



197

µm × 0.5 µm film thickness. The temperature was programmed to increase from 120

198

to 180 °C at a rate of 12 °C per min and continues to keep 1 min, after the

199

programmed to increase from 180 to 250 °C at a rate of 8 °C per min and continues to

200

keep 15 min. FA in samples was identified by library searching of NIST. The relative

201

amount of each FA was calculated from their peak areas as compared with that of

202

standards.

203

Analysis of root tip surface and root meristem of B. pilosa treated with

204

berberine.

205

Scanning electron microscopy (SEM) was performed in order to examine the

206

effect of berberine on the root surface of 7 d old B. pilosa seedlings, treated with

207

berbeine at 3.2 mg L−1 and 32 mg L−1 respectively. The SEM samples were processed

208

as previously described by Cao et al.26 Root tip samples were visualized by SEM (FEI

209

XL-30, Netherlands).

210

Transmission electron microscopy (TEM) was performed to examine the effect

211

of berberine on root meristem cells of 7 d old B. pilosa seedlings, treated berberine at

212

3.2 mg L−1 and 32 mg L−1. The TEM samples were processed as previously described

213

by Houot et al.27 Root tip samples were examined by TEM (FEI Tecnai 12,

214

Netherlands).

215

Safety evaluation of berberine on sensitive organism, Brachydanio rerio

216

Brachydania rerio was domesticated in a tank of dechlorinated water for 1 week

217

before the start of the experiment. During domestication, B. rerio were fed once per

218

day. Fasting was forced upon them before the day of experiment. The death rate was

10


Page 10 of 51

Page 11 of 51


219

below 1% during the domestication period. The B. rerio length was 3.05±0.26 cm,

220

and the corresponding weight was 0.26±0.06 g. The acute toxicity was measured by

221

the method of Botelho et al.28

222

Statistical analysis

223

The inhibition rates of the fresh weight or the root lengths of tested weeds were

224

analyzed using SPSS 19.0 with the probit analysis. Then, the estimation of the median

225

inhibition concentration (IC50) or the median lethal concentration (LC50) and its 95%

226

confidence limits were obtained. All quantitative data were presented as the mean ±

227

SD of at least three independent experiments using the Duncan's multiple range test or

228

Student’s t test for group differences. A p < 0.05 was considered as statistically

229

significant.

230

RESULTS AND DISCUSSION

231

Herbicidal activity of berberine

232

Berberine displayed broad spectrum herbicidal activities against a variety of

233

weeds including monocots and dicots. However, berberine at 2-32 mg L−1 showed no

234

effect on the germination of these tested plants (Table 1).

235

The IC50 values of berberine on the primary root length of dicot plants, Ageratum

236

conyzoides, Mikania micrantha, Celosia argentea Amaranthus retroflexus and Bidens

237

pilosa 7 d after the treatment, were 2.91, 2.36, 2.39 3.76 and 3.03 mg L−1, respectively,

238

and the IC50 values of berberine on fresh weight of Ageratum conyzoides, Mikania

239

micrantha, Celosia argentea Amaranthus retroflexus and Bidens pilosa were 5.76,

240

6.42, 5.83, 4.57 and 6.45 mg L−1, respectively. In parallel, the IC50 values of berberine

11



241

on root lengths of monocot plants, Echinochloa crusgalli, Dactyloctenium willd,

242

Eleusine indica, Triticum aestivum and Oryza sativa 7 d after the treatment were 6.71,

243

5.44, 4.83, 9.79 and 6.79 mg L−1, respectively, and the IC50 values of berberine on

244

fresh weight of Echinochloa crusgalli, Dactyloctenium willd, Eleusine indica,

245

Triticum aestivum and Oryza sativa 7 d after the treatment were 16.89, 18.83, 17.32,

246

28.36 and 35.07 mg L−1, respectively, which were only slightly higher than those of

247

dicot plants, indicating that berberine has non-selective herbicidal activities among

248

monocot and dicot weeds (Table2, Table 3). The selectivity of berberine is similar to

249

other non-selective bioherbicides.29

250

The herbicidal activity of berberine on L. minor was examined with 10 mg L−1

251

berberine. Between 3 to 14 d, the biomass of untreated L. minor increased 17.32 times

252

(181.90 mg /10.50 mg), and meanwhile their thallus number also increased 5.17 times

253

((131.67-21.33) /21.33). By contrast, the treated L. minor decreased 6.22 times

254

(-16.8/-2.7) in biomass, and produced no new thallus (Table 4). Especially, 3 d after

255

treatment, L. minor turned to yellow; when the treatment was extended to 9 d, all of

256

the L. minor plants have rotted completely (Fig.2). The yellowish phenotype in treated

257

L. minor suggested that chlorophyll synthesis could be inhibited. Indeed, after 3 d,

258

treatment, chlorophyll a and b were reduced to 52.24% and 50.53% of untreated L.

259

minor, respectively. After 7 d, the synthesis of chlorophyll a, chlorophyll b and the

260

total chlorophyll were all completely abolished (Table 5, Table 6). These results

261

indicated that 10 mg L−1 berberine possessed significant herbicidal activity on L.

262

minor, which is not consistent with what was reported by Iwasa (2000), who showed

12


Page 12 of 51

Page 13 of 51


263

that 10 mg L−1 berbeine only resulted in a 75% inhibition rate and therefore berbeine

264

has no potential for agrochemical use (Iwasa, 2000).15 This inconsistency could be

265

resulted from the different growth potentials of different L. minor populations and the

266

different test temperatures.

267

In a 14-d treatment, berberine reduced fresh weight of B. pilosa by 80.91% at 16

268

mg L−1; and by 84.84% at 32 mg L−1. Similar reduction was observed with

269

cinmethylin treatment (82.62% at 16 mg L−1; and 86.24% at 32 mg L−1). Fresh weight

270

loss caused by glyphosate in the similar condition was 93.66% at 16 mg L−1; and

271

95.56% at 32 mg L−1, significantly higher than that caused by either berberine or

272

cinmethylin (Table 7). In the concentration between 4 to 32 mg L−1, berberine

273

inhibited B. pilosa primary root growth with a rate similar to that of cinmethylin and

274

there were no significant differences. However, generally, glyphosate showed better

275

efficacy to B. pilosa those of berberine and cinmethylin (Table 7). This is very active

276

for a natural product and QSAR of berberine and its possible derivatives need to be

277

further studied.

278

Sulcotrione and 2,4-D both showed the highest herbicidal activity on B. pilosa

279

among the five weeds we tested here. They were found to inhibit the germination

280

process, as shown in figure 3, therefore the inhibition of fresh weight and the primary

281

root were not calculated.

282

More often, natural herbicidal compounds are considered as lead compound due

283

to their less potent activity compared with their synthetic counterparts (glufosinate

284

and the triketone herbicides).3 Nevertheless, they still have great potential in their

13



285

possible new mode of action, a consideration needed in order to deal with the

286

evolution of resistance to herbicides in agricultural production. For example, the

287

optimal concentration of sarmentine, isolated from Piper longum, for excellent control

288

of barnyard grass was 5 mg/mL.4 Still, sarmentine was attractive due to its multiple

289

herbicide mechanisms of action.30 Surely scientists have found some plant-derived

290

compounds with distinctive herbicidal activity such as patented m-tyrosine.31 In our

291

study, 7 d after the treatment, the IC50 of berberine, glyphosate and 2,4-D on the root

292

growth of B. pilosa were 3.27, 3.45 and 0.23 mg L−1, respectively, whereas the IC50

293

values of berberine, glyphosate and 2,4-D on the fresh weight of B. pilosa were 6.11,

294

7.72 and 0.92 mg L−1, respectively.

295

In conclusion, berberine possessed broad spectrum herbicidal activities on both

296

moncots and dicots and showed similar effect on B. pilosa to the commercial naturally

297

derived herbicide cinmethylin. These results indicated that berberine could be used as

298

a new bioherbicide directly or serve as a template compound for a new type of

299

herbicides.

300

Absorption and transport of berberine in B. pilosa.

301

Berberine treatment caused a continuous increase in the inhibition rates (the

302

inhibition rate of root elongation, whole plant height, fresh root weight and fresh plant

303

weight) in root incubation treatment during the time intervals from 1 to 7 d. However,

304

the inhibition rates of root elongation, plant height, fresh root weight and fresh plant

305

weight caused by both stem-coating and leaf-dipping methods were not significant,

306

which indicated that berberine was mainly absorbed by root (Fig.4).

14


Page 14 of 51

Page 15 of 51


307

To examine the absorption and transport pattern of berberine in the plant, B.

308

pilosa seedlings (incubated in Hoagland solution for 50 d) were treated by root

309

incubation in a water solution of berberine at a concentration of 32 mg L−1, or by stem

310

coating with berberine at a concentration of 1000 mg L−1, or by leaf painting with

311

berberine at a concentration of 1000 mg L−1.

312

After berberine treatment (32 mg L−1) by root incubation, the accumulation of

313

berberine in stem, leaf and root were detected by HPLC at different time points.

314

During the time periods of 0.5, 1, 2, 3, 5 and 7 d after treatment, the concentrations of

315

berberine in roots were 51.69, 63.59, 76.74, 50.45, 38.33 and 27.38 µg g-1,

316

respectively; and were 6.41, 8.05, 12.33, 19.23, 14.58 and 9.09 µg g-1, respectively in

317

stems; and were 0, 0, 2.76, 3.17, 3.14 and 2.63 µg g-1, respectively in leaves (Fig. 5A).

318

These results indicate that berberine was gradually absorbed by root and was

319

transported upward to stem and leaf. As it was shown (Fig. 5A), berberine

320

concentration in root reached peak at 2 d after the treatment due to the absorption and

321

accumulation of berberine at the first 2 d and decreased after 2 d, suggesting that

322

berberine degradation occurred 2 d after the treatment. Berberine concentration in the

323

root reached a peak at 3 d after the treatment in stem and leaf and decreased

324

afterwards (Fig 5A), possibly due to the relatively long distance of transport from root

325

to leaf.

326

The rate of degradation of berberine in the incubation solution with or without B.

327

pilosa were different. Without B. pilosa, 0.5, 1, and 2 d after the treatment, berberine

328

concentrations remaining in the solution were 31.99, 31.53 and 25.54 mg L−1,

15



329

respectively; no berberine could be detected after 3 d of the treatment. Interestingly,

330

with B. pilosa, 0.5, 1, 2 and 3 d after the treatment, berberine remaining in the

331

solution were 30.96, 28.96, 26.69 and 13.73 mg L−1, while berberine could not be

332

detected 5 d after the treatment. The results indicated that berberine was degraded

333

faster in the control than in the treatment. The difference in the degradation rates

334

between the control and the treatment might be due to the effect of existence of the

335

root exudate of B. pilosa on berberine (Fig.5B).

336

After berberine treatment (1000 mg L−1) by leaf painting, berberine

337

concentrations in leaves during the time periods of 0.5, 1, 2, 3, 5 and 7 d, were 14.01,

338

18.07, 28.31, 18.86, 13.94 and 9.30 µg g-1, respectively (Fig. 5C). However, berberine

339

was not detectable in stems and roots (Fig. 5C). This result suggested that berberine

340

was transported acropetally, but not basipetally.

341

The uptake and transport of berberine within B. pilosa seedlings was visualized

342

under the fluorescence microscope. In roots, we observed that fluorescence was

343

present in endodermis, pericycle, phloem and protoxylem in the triarch root of B.

344

pilosa 0.5, 1, 2, 3 and 5 d after the treatment of berberine at 32 mg L−1. In the lower

345

part of the stem, with increasing times of treatment (0.5, 1, 2, 3 and 5 d) fluorescence

346

became more prominent in vascular bundles. From row 2 to 5, we observed quick

347

absorption and transport of berberine in root and the gradual transport and

348

accumulation of berberine through the vascular bundle in stem to leaves (Fig. 6).

349 350

In conclusion, berberine was mainly absorbed by plant root and was acropetally transported through vascular bundle in stem to leaves.

16


Page 16 of 51

Page 17 of 51


351

Effects of berberine on physiological activities of B. pilosa.

352

Roots play an important role in the nutrient cycling of terrestrial ecosystems

353

because they constitute both sinks and sources for nutrients. Berberine caused the

354

increase of root vigor at concentrations ranging from 4 to 32 mg L−1, reaching a peak

355

at 16 mg L−1 berberine and the root vigor of which was 6.6 times of the control (Fig.

356

7A). This could be due to the increasing amount of berberine induced stress on B.

357

pilosa root and thus stimulated the metabolic activities in related root cells to degrade

358

the alien toxin, berberine. However, berberine at concentrations higher than 16 mg L−1

359

might cause an irreversible phytotoxic effect on root cells and therefore induced a

360

decrease of root vigor.

361

Environmental stresses, including herbicidal stress, induced the production of

362

reactive oxygen species (ROS), which can cause oxidative damage to plant cells.32

363

The responses of the antioxidant enzymes to berberine stress were different (Fig. 7).

364

The induction of POD activity in plants occurs in response to numerous biotic and

365

abiotic stimuli, including exposure to pathogens or elicitor preparations, chemical

366

oxidizing agents, red light, and mechanical stimuli. The roles that POD can play in

367

strengthening the cell wall and in the production of toxic secondary metabolites and

368

its simultaneous oxidant and anti-oxidant capabilities can make it an important factor

369

in the integrated defense response of plants to a variety of stresses.33 PPO catalyzes

370

the oxygen-dependent oxidation of mono- and o-diphenols to o-diquinones, highly

371

reactive intermediates, the secondary reactions of which are believed to be responsible

372

for the oxidative browning that occurs as a consequence of plant senescence,

17



373

wounding and pathogen infection.34 PPOs have been known to biochemistry for a

374

century and several hypotheses regarding the function of PPO have been proposed,

375

including roles in the phenylpropanoid pathway and plant defense.35 A defensive role

376

for PPO has frequently been suggested due to the conspicuous appearance of PPO

377

reaction products upon wounding, pathogen infection, or insect infestation, and due to

378

the inducibility of PPO in response to various abiotic and biotic injuries or signaling

379

molecules.36 Berberine treatment caused almost a continuous increase in the POD and

380

PPO activities as berberine concentrations increased from 8 to 32 mg L−1 (Fig. 7B),

381

suggesting that the exposure to berberine caused an obvious oxidative damage to B.

382

pilosa. Berberine increased the POD and PPO activities more significantly at higher

383

concentrations. POD activities were 3.1, 2.6 and 11.4 times higher than the control

384

with the treatment of berberine at 8, 16 and 32 mg L−1, respectively, and PPO

385

activities were 3.1, 2.6 and 11.4 times higher than the control with the treatment of

386

berberine at 8, 16 and 32 mg L−1, respectively.

387

As antioxidative enzymes in plants, SOD plays important roles in scavenging

388

ROS produced under oxidative stress. The reduction of the enzyme activities can be

389

considered as indirect evidence for enhanced production of ROS.37 The activities of

390

SOD in B. pilosa (Fig. 7B) were obviously inhibited by berberine, and the inhibition

391

rates increased with increasing berberine concentrations. Berberine treatment had no

392

significant impact on SOD activity at the concentration of 4 mg L−1. However,

393

berberine at concentrations from 8 to 32 mg L−1 caused decreases in SOD activity.

394

Antioxidant enzymes are important for the metabolism of xenobiotics.38 These

18


Page 18 of 51

Page 19 of 51


395

enzymes can be inactivated under a severe oxidative stress.39 We speculate that the

396

inhibition of SOD might be due to the accumulation of ROS induced by berberine.

397

The MDA accumulation is an indicator of lipid peroxidation in plant cells.40 We

398

measured MDA levels to quantify lipid peroxidation. Berberine treatments at

399

concentrations of 4 to 32 mg L−1 significantly increased the levels of MDA in B.

400

pilosa root (Fig. 7C). The MDA accumulation reached a maximum at berberine

401

concentration of 8 mg L−1 (about 48 nmol•gFW-1 in root) and remained at

402

concentrations of up to 32 mg L−1, indicating a higher degree of lipid peroxidation due

403

to berberine stress. Our results from the antioxidant enzyme activities and MDA

404

content indicated that berberine induced the oxidative stress and a higher

405

concentration of berberine could cause a stronger oxidative damage to B. pilosa.

the

406

Lipid peroxidation alters membrane properties and causes cell defects such as ion

407

leakage and cellular decompartmentation.41-42 Fatty acid composition can affect the

408

membrane fluidity, stability, and functions. The main roles of fatty acids in plant are

409

related to cell membrane functions and to metabolic processes. The degree of fatty

410

acid unsaturation is important in maintaining the fluidity of the membrane and in

411

providing the appropriate environment for membrane functions. The degree of

412

unsaturation of membrane fatty acids is also important in the process of plant

413

adaptation to growth environment.43 The computation of the index of unsaturated fatty

414

acids (IUFA) was calculated according to Liu et al.44 The results showed that the

415

IUFA first increased with berberine at 12.5 to 50 mg L−1 and then decreased with

416

berberine at 50 to 200 mg L−1 (Fig. 7D), suggesting lower concentration of berberine

19



417

caused a positive reaction of B. pilosa to adapt to the stress by berberine treatment.

418

Nevertheless, higher concentrations of berberine from 50 to 200 mg L−1 caused

419

irreversible changes, revealed by the continuous decrease of IUFA percent. The

420

degree of fatty acid unsaturation is the more important factor intervening in the

421

maintaining of membrane fluidity and provides the appropriate environment for

422

membrane function and the processes of plant adaptation in the face of constraining

423

conditions. The plasma membrane is at the interface between the cell and its

424

environment, and serves to hold intact the entire cellular structure. Consequently,

425

destabilization of the lipid bilayer, often via reactive oxygen species (ROS)-induced

426

stress membrane peroxidation, results in uncontrolled electrolyte leakage and cell

427

death.45 Our results suggested that berberine disrupt the IUFA percent of cell

428

membrane and therefore affect membrane fluidity, which was eventually expressed as

429

decrease of adaptation ability of cell to stress environment and abnormal cell function

430

or even cell death.

431

Alteration of the root surface ultrastructure of B. pilosa by berberine

432

SEM images of the root surface of B. pilosa were acquired at magnifications of

433

800× and 12800× to study the effect of berberine on root inhibition of B. pilosa (Fig.

434

8).

435

Under a magnification of 800×, we observed regular longitudinal ridges on the

436

root surface in both control samples and samples treated with lower concentration of

437

berberine (3.2 mg L−1). However, the regular longitudinal ridges on the root surface

438

almost disappeared and irregular unevenness appeared with the treatment of higher

20


Page 20 of 51

Page 21 of 51


439

concentration of berberine (32 mg L−1) (Fig. 8A, B, C).

440

Under higher magnification of 12800×, we observed smooth root surfaces in

441

control samples. By contrast, obvious shrinking occurred on the root surface with

442

berberine at 3.2 mg L−1. In addition to the shrinkage we also observed uneven

443

projections on the root surface when treated with berberine at 32 mg L−1 (Fig. 8D, E,

444

F).

445

These morphological changes indicated that berberine treatment might cause

446

damage in subcellular organization to root cells. Therefore, we conducted SEM

447

analysis to investigate the subcellular effect of berberine on root cells.

448

Longitudinal sections of root cells treated with 3.2 or 32 mg L−1 berberine for 7 d

449

were examined by conventional electron microscopy. TEM analysis of the

450

longitudinal sections of root cells showed chromatin condensation in nucleus,

451

nucleolus condensation, mitochondria collapse, and a slight increase in cell wall

452

opacity when plants were treated with 3.2 mg L−1 berberine. Severe damage was

453

observed in cell structure at higher berberine concentration 32 mg L−1. In this

454

treatment, plasmolysis occurred and damage to basic cell structure was severe, as

455

revealed by the fact that nucleolus condensation became more apparent, and as well

456

by damage to both the nuclear envelop and the plasma membrane and was

457

accompanied by increased cell wall opacity. As a contrast, intact nuclear envelope and

458

normally distributed chromatin were present in the control roots.

459

The appearance of invaginations of the nuclear envelope was also observed in

460

the treatment groups. Images of the nuclear bulge showed that these membrane

21



461

profiles originated from the invaginations of the nuclear envelope. At these late stages

462

the nucleus showed a high condensation of the remaining chromatin and numerous

463

vesicles (Fig. 9E). These cells present anarchic structural changes and resemble

464

explosive necrosis.

465

apoptotic-like cell death.

466

Safety evaluation of berberine to aquatic organisms

27

Hence, berberine treatment appeared to induce necrosis and

467

The LC50 of dichromate to the tested batch of B. rerio was 241.32 mg L−1, which

468

met the required LC50 values of dichromate on the tested B. rerio at 24 h between 200

469

and 400 mg L−1. The LC50 value of berberine to B. rerio 96 h after the treatment were

470

126.38 mg L−1, which revealed that berberine was low toxic to B. rerio (Table 8). As a

471

contrast, the 96 h LC50 of commercially used herbicide atrazine to B. rerio is 29.06

472

mg L−1.46

473

Generally, berberine is considered to be a non-toxic alkaloid at doses used in

474

clinical situations.47 A dose up to 14,500 mg L−1 in rats and 7,000 mg L−1 in mice did

475

not cause any maternal deaths. The lowest observed adverse effect levels in maternal

476

rats and developmental toxicity LOAEL, based on reduced fetal body weight per litter

477

was 7,250 mg L−1 and 14,500 mg L−1, respectively. There were no fetal adverse

478

effects noted.48 Briefly, berberine was low toxic to the sensitive organism,

479

Brachydanio rerio. Therefore, the use of berberine as a safe, biocontrol agent on

480

weeds is promising.

481 482

AUTHOR INFORMATION 22


Page 22 of 51

Page 23 of 51


483

Corresponding Author

484

*（L.-J.Z.）Phone: 86 15917363981. E-mail: [email protected].

485

ORCID

486

Jiao Wu: 0000-0003-1871-6034

487

Lijuan Zhou: 0000-0002-7720-4138

488

Author Contributions

489

||

490

Funding

491

This work was supported by Science and Technology Planning Programs of

492

Guangdong Province, China (2016A050502047 and 2015A020209151) and Science

493

and Technology Program of Guangzhou, China (201607010125)

494

Notes

495

The authors declare no competing financial interest.

J.W and JJ.M contributed equally to this work.

496

497

ABBREVIATIONS USED

498

ALS, acetolactate synthase; AR, analytical reagent; FA, fatty acid; GC-MS, gas

499

chromatography-mass spectrometry; IUFA, index of unsaturated fatty acids; MDA,

500

malondialdehyde; POD, peroxidase; PPO, polyphenol oxidase; ROS, reactive oxygen

501

species; SEM, scanning electron microscope; SOD, superoxide dismutase; TBA,

502

thiobarbituric acid; TBARS, 2-thiobarbituric acid reactive substances; TCA,

503

trichloroacetic acid; TEM, transmission electron microscopy; TTC, triphenyl

504

tetrazolium chloride.

505 506

ACKNOWLEDGMENT 23



507

We thank all members of the department of Pesticide Science, South China

508

Agricultural University for the help with this project. This project was funded by

509

Science and Technology Planning Programs of Guangdong Province, China

510

(2016A050502047 and 2015A020209151) to L. Z. and Science and Technology

511

Program of Guangzhou, China (201607010125) to L. Z..

512 513

REFERENCES

514

(1) Dayan, F. E.; Owens D. K.; Duke, S. O. Rationale for a natural products approach to herbicide

515 516 517 518 519

discovery. Pest Manag. Sci. 2012, 68, 519-528. (2) Dayan, F. E.; Duke, S. O. Natural compounds as next-generation herbicides. Plant Physiol. 2014, 166, 1090-1105. (3) Cantrell, C. L.; Dayan, F. E.; Duke, S. O. Natural products as sources for new pesticides. J. Nat. Prod. 2012, 75, 1231-1242.

520

(4) Huang, H.; Morgan, C. M.; Asolkar, R. N.; Koivunen, M. E.; Marrone, P. G. Phytotoxicity of

521

sarmentine isolated from long pepper (Piper longum) fruit. J. Agric. Food Chem. 2010, 58,

522

9994-10000.

523 524 525 526

(5) Dayan, F. E.; Cantrell, C. L.; Duke, S. O. Natural products in crop protection. Bioorg. Med. Chem. 2009, 17, 4022- 4034. (6) Vyvyan, J. R. Allelochemicals as leads for new herbicides and agrochemicals. Tetrahedron. 2002, 58, 1631-1646.

527

(7) Imperatore, C.; Aiello, A., D'Aniello, F.; Senese, M.; Menna, M. Alkaloids from marine

528

invertebrates as important leads for anticancer drugs discovery and development. Molecules. 2014, 19,

529

20391-20423.

530

(8) Iqbal, Z.; Hiradate, S; Noda, A. Allelopathy of buckwheat: assessment of allelopathic potential of

531

extract of aerial parts of buckwheat and identification of fagomine and other related alkaloids as

532

allelochemicals. Weed Biol. Manage. 2002, 2, 110-115.

533

(9) Kumar, A.; Ekavali; Chopra, K.; Mukherjee, M.; Pottabathini, R.; Dhull, D. K. Current knowledge

24


Page 24 of 51

Page 25 of 51


534 535 536 537 538 539 540 541 542

and pharmacological profile of berberine: An update. Eur. J. Pharmacol. 2015, 761, 288-297. (10) Miyazawa, M.; Fujioka, J.; Ishikawa, Y. Insecticidal compounds from Phellodendron amurense active against Drosophila melanogaster. J. Sci. Food Agric. 2002, 82, 830-833. (11) Hou, D.; Yan, C.; Liu, H.; Ge, X.; Xu, W.; Tian, P. Berberine as a natural compound inhibits the development of brown rot fungus Monilinia fructicola. Crop Prot. 2010, 29, 979-984. (12) Zhou, L. J.; Huang, J. G.; Xu, H. H. Herbicidal use of berberine. CPB. No. 201110245670.6. 2012-02-15. (13) Zhou, L. J.; Huang, J. G.; Sun, Y, Y. Herbicidal activities of methanol extracts of 29 plants against Pistia stratiotes L. J. Huazhong Agric. Univ. 2011, 30, 200-205.

543

(14) Li, W.; An, P.; Liu, X. Effect of storage, stratification, temperature and gibberellins on

544

germination of dimorphic seeds of Suaeda salsa under saline conditions. Seed Sci. Technol. 2008, 36,

545

122-132.

546 547

(15) Iwasa, K.; Moriyasu, M.; Nader, B. Fungicidal and herbicidal activities of berberine related alkaloids. Biosci., Biotechnol., Biochem. 2000, 64, 1998-2000.

548

(16) Rashid, M. H.; Asaeda, T.; Uddin, M. N. Litter-mediated allelopathic effects of kudzu (Pueraria

549

montana) on Bidens pilosa and Lolium perenne and its persistence in soil. Weed Biol. Manage. 2010,

550

10, 48-56.

551

(17) Kamal, Y. T.; Singh, M.; Tamboli, E. T.; Parveen, R.; Ahmad, S. Quantitative analysis of

552

berberine in Berberis aristata fruits and in a traditional anti-inflammatory unani formulation by use of

553

a validated HPLC method. Acta Chromatogr. 2011, 23, 157-168.

554

(18) Li, D.; Xu, Y.; Zhang, D.; Quan, H.; Mylonakis, E.; Hu, D.; Li, M.; Zhao, L.; Zhu, L.; Wang, Y.;

555

Jiang, Y. Fluconazole assists berberine to kill fluconazole-resistant Candida albicans. Antimicrob.

556

Agents Chemother. 2013, 57, 6016-6027.

557 558 559 560

(19) Wang, J.; Lei, Z.; Wen, Y.; Mao, G.; Wu, H.; Xu, H. A novel fluorescent conjugate applicable to visualize the translocation of glucose-fipronil. J. Agric. Food Chem. 2014, 62, 8791-8798. (20) Ruf, M.; Brunner, I. Vitality of tree fine roots: reevaluation of the tetrazolium test. Tree Physiol. 2003, 23, 257-263.

561

(21) Zhang, H.; Xia, Y.; Wang, G.; Shen, Z. Excess copper induces accumulation of hydrogen

562

peroxide and increases lipid peroxidation and total activity of copper-zinc superoxide dismutase in

563

roots of Elsholtzia haichowensis. Planta. 2008, 227, 465-475. 25



564 565 566 567

(22) Thipyapong, P.; Melkonian, J.; Wolfe, D. W.; Steffens, J. C. Suppression of polyphenol oxidases increases stress tolerance in tomato. Plant Sci. 2004, 167, 693-703. (23) Zipor, G.; Oren-Shamir, M. Do vacuolar peroxidases act as plant caretakers? Plant Sci. 2013, 199, 41-47.

568

(24) Hajlaoui, H.; Denden, M.; El Ayeb, N. Changes in fatty acids composition, hydrogen peroxide

569

generation and lipid peroxidation of salt-stressed corn (Zea mays L.) roots. Acta Physiol. Plant. 2009,

570

31, 787-796.

571 572

(25) Bettaieb, I.; Zakhama, N.; Wannes, W. A.; Kchouk, M. E.; Marzouk, B. Water deficit effects on Salvia officinalis fatty acids and essential oils composition. Sci. Hortic. 2009, 120, 271-275.

573

(26) Cao, X.; Chen, C.; Zhang, D.; Shu, B.; Xiao, J.; Xia, R. Influence of nutrient deficiency on root

574

architecture and root hair morphology of trifoliate orange (Poncirus trifoliata L. Raf.) seedlings under

575

sand culture. Sci. Hortic. 2013, 162, 100-105.

576

(27) Houot, V.; Etienne, P.; Petitot, A.; Barbier, S.; Blein, J.; Suty, L. Hydrogen peroxide induces

577

programmed cell death features in cultured tobacco BY-2 cells, in a dose-dependent manner. J. Exp.

578

Bot. 2001, 52, 1721-1730.

579

(28) Botelho, R. G.; Dos Santos, J. B.; Fernandes, K. M.; Neves, C. A. Effects of atrazine and

580

picloram on grass carp: acute toxicity and histological assessment. Toxicol. Environ. Chem. 2012, 94,

581

121-127.

582 583

(29) Fukuda, M.; Tsujino, Y.; Fujimori, T.; Wakabayashi, K.; Böger, P. Phytotoxic activity of middle-chain fatty acids I: effects on cell constituents. Pestic. Biochem. Physiol. 2004, 80, 143-150.

584

(30) Dayan, F. E.; Owens, D. K.; Watson, S. B.; Asolkar, R. N.; Boddy, L. G. Sarmentine, a natural

585

herbicide from Piper species with multiple herbicide mechanisms of action. Front. Plant Sci. 2015, 6,

586

222. doi: 10.3389/fpls.2015.00222.

587

(31) Bertin, C.; Weston, L. A.; Huang, T.; Jander, G.; Owens, T.; Meinwald, J.; Schroeder, F. C.

588

Grass roots chemistry: meta-Tyrosine, an herbicidal nonprotein amino acid. Proc. Natl. Acad. Sci. U. S.

589

A. 2007, 104, 16964-16969.

590 591

(32) Song, N. H.; Yin, X. L.; Chen, G. F.; Yang, H. Biological responses of wheat (Triticum aestivum) plants to the herbicide chlorotoluron in soils. Chemosphere. 2007, 68, 1779-1787.

592

(33) Hammerschmidt, R.; Nuckles, E. M.; Kuć, J. Association of enhanced peroxidase activity with

593

induced systemic resistance of cucumber to Colletotrichum lagenarium. Physiol. Plant Pathol. 1982, 26


Page 26 of 51

Page 27 of 51


594

20, 73- 82.

595

(34) Casal, J. J.; Mella, R. A.; Ballare, C. L.; Maldonado, S. Phytochrome-mediated effects on

596

extracellular peroxidase activity, lignin content and bending resistance in etiolated Vicia faba epicotyls.

597

Physiol. Plant. 1994, 92, 555-562.

598 599 600 601 602 603 604 605

(35) Thipyapong, P.; Stout, M. J.; Attajarusit, J. Functional analysis of polyphenol oxidases by antisense/sense technology. Mol. 2007, 12, 1569-1595. (36) Kojima, M.; Takeuchi, W. Detection and characterization of p-coumaric acid hydroxylase in mung bean, Vigna mungo, seedlings. J. Biochem. 1989, 105, 265-70. (37) Wen, Y.; Chen, H.; Shen, C.; Zhao, M.; Liu, W. Enantioselectivity tuning of chiral herbicide dichlorprop by copper: roles of reactive oxygen species. Environ. Sci. Technol. 2011, 45, 4778-4784. (38) Liu, H.; Huang, R.; Xie, F.; Zhang, S.; Shi, J. Enantioselective phytotoxicity of metolachlor against maize and rice roots. J. Hazard. Mater. 2012, 217, 330-337.

606

(39) Sun, Y.; Guo, H.; Yu, H.; Wang, X.; Wu, J.; Xue, Y. Bioaccumulation and physiological effects

607

of tetrabromobisphenol A in coontail Ceratophyllum demersum L. Chemosphere. 2008, 70, 1787-1795.

608

(40) Meloni, D. A.; Oliva, M. A.; Martinez, C. A.; Cambraia, J. Photosynthesis and activity of

609

superoxide dismutase, peroxidase and glutathione reductase in cotton under salt stress. Environ. Exp.

610

Bot. 2003, 49, 69-76.

611 612 613 614 615 616 617 618

(41) Blokhina, O.; Virolainen, E.; Fagerstedt, K. V. Antioxidants, oxidative damage and oxygen deprivation stress: a review. Ann. Bot. 2003, 91, 179-194. (42) Yang, Z.; Liu, C.; Xiang, L.; Zheng, Y. Phenolic alkaloids as a new class of antioxidants in Portulaca oleracea. Phytother. Res. 2009, 23, 1032-1035. (43) Xu, X.; Beardall, J. Effect of salinity on fatty acid composition of a green microalga from an Antarctic hypersaline lake. Phytochemistry. 1997, 45, 655-658. (44) Liu, S. H.; Chen, G. X.; Yin, J. J.; Lu, C. G. Response of the flag leaves of a super-hybrid rice variety to drought stress during grain filling period. J. Agron. Crop Sci. 2011, 197, 322-328.

619

(45) Mittler, R. Oxidative stress, antioxidants and tolerance. Trends Plant Sci. 2002, 7, 405-410.

620

(46) Yan, H.; Huang, S.; Scholz, M. Kinetic processes of acute atrazine toxicity to Brachydanio rerio

621

in the presence and absence of suspended sediments. Water, Air, Soil Pollut. 2015, 226, 1-13.

622

(47) Rabbani, G. H.; Butler, T.; Knight, J.; Sanyal, S. C.; Alam, K. Randomized controlled trial of

623

berberine sulfate therapy for diarrhea due to enterotoxigenic Escherichia coli and Vibrio cholerae. J. 27



624

Infect. Dis. 1987, 155, 979-84.

625

(48) Jahnke, G. D.; Price, C. J.; Marr, M. C.; Myers, C. B.; George, J. D. Developmental toxicity

626

evaluation of berberine in rats and mice. Birth Defects Research Part B-developmental and

627

Reproductive Toxicology. 2006, 77, 195-206.

628 629 630 631 632 633 634 635 636 637 638 639 640 641 642 643 644 645 646 647 648 649 650 651 652

28


Page 28 of 51

Page 29 of 51


653

Figure 1. Molecular structure of berberine.

654

Figure 2. Inhibitory effect of 10 mg L-1 berberine on L. minor. (A): Overall view of L. minor; (B):

655

Change of biomass; (C): Change of thallus number; (D) Chlorophyll a content; (E): Chlorophyll b

656

content; (F): Total chlorophyll content; (G): Inhibition rate of chlorophyll content. Letters represent

657

values that differed significantly in the Student’s t test (A,B,C,D,E,F) and Duncan's multiple range test

658

(G) (P < 0.05). Shown are the averages of 30 plants ± SE (n = 3)

659

Figure 3. Inhibitory effect of berberine, glyphosate, cinmethylin, sulcotrione and 2,4-D at different

660

concentration on B. pilosa. (A): Overall view of B. pilosa; (B): Inhibition rate of fresh weight; (C):

661

Inhibition rate of primary root length. Letters represent values that differed significantly in the

662

Duncan's multiple range test (P < 0.05). Shown are the averages of 30 plants ± SE (n = 3)

663

Figure 4. Inhibitory effect of berberine with different application methods (root incubation,

664

stem-coating and leaf-dipping ) on B. pilosa. (A): Root inhibition rate; (B): Plant height inhibition rate;

665

(C): Fresh root weight inhibition rate; (D): Total fresh plant weight inhibition rate. Letters represent

666

values that differed significantly in the Duncan's multiple range test (P < 0.05). Shown are the averages

667

of 30 plants ± SD (n = 3)

668

Figure 5. Uptake of berberine by root incubation and leaf coating obtained from B. pilosa

669

seedlings. Data are mean of 9 plant ± SE (n = 3). (A) Distribution of berberine in B. pilosa with

670

treatment of 32 mg L−1. (B) The remaining amount of berberine in water solution and control, shown

671

are the averages ± SE (n = 3). (C) Distribution of berberine with leaf coating at 1000 mg L−1

672

concentration of 40µL, each bar represents the mean of 9 plants ± SE, n = 3.

673

Figure 6. Representative fluorescence photographs of berberine in 6-d-old B. pilosa seedlings: The

674

roots were incubated in a solution containing 32 mg L−1 berberine. A1, B1, C1, D1, E1 and F1 was root;

29



675

A2, B2, C2, D2, E2 and F2 was the lower part of the stem; A3, B3, C3, D3, E3 and F3 was the upper

676

stem; A4, B4, C4,D4, E4 and F4 was the first true leaf petiole; A5, B5, C5,D5, E5 and was the second

677

true leaf petiole; A (A1, A2, A3, A4 and A5), B (B1, B2, B3, B4 and B5), C (C1, C2, C3, C4 and C5),

678

D (D1, D2, D3, D4 and D5), E (E1, E2, E3, E4 and E5) and F (F1, F2, F3, F4 and F5) were control, 0.5,

679

1, 2, 3, 5 and 7 d, respectively. The presence of fluorescent berberine was indicated by arrowheads. P:

680

pith. VB: vascular bundle.

681

Figure 7. Physiological effects of berberine on B. pilosa. A: Effects of berberine on root vigor of B.

682

pilosa root; B: Effects of berberine on POD, PPO and SOD activity of B. pilosa root; C: Effects of

683

berberine on MDA of B. pilosa root; D: Effect of berberine on IUFA of B. pilosa. Letters represent

684


685

± SD (n = 3). Index of unsaturated fatty acid (IUFA): {[16 : 1 %×1] + [18 : 1 % ×1] + [18 : 2 %×2]

686

+ [18 : 3 %×3]} ×100 % .

687 688

47

Figure 8. SEM analysis of B. pilosa root surface under control (A and D) and treatment (B, E and C, D) with berberine (3.2 and 32 mg L−1)

689

Figure 9. Ultrastructure changes observed in root cells treated with berberine. Root cells were

690

treated with 3.2 and 32 mg L−1 berberine for 7 d, observed by transmission electron microscopy. TEM

691

details of the effect of berberine. The control cells (A, B, C), treatment with berberine (D, E, F treated

692

with berberine 3.2 mg L−1 and G, H, I treated with berberine at 32 mg L−1).

693 694 695 696 697 698 699 700

30


Page 30 of 51

Page 31 of 51


701

Table 1 The effect of berberine on the germination rates of some plants

Test plant

Ageratum conyzoides

Mikania micrantha

Germination rate (%)

Time (d)

Control

2 mg L-1

4 mg L-1

8 mg L-1

16 mg L-1

32 mg L-1

1

0.00±0.00a

0.00±0.00a

0.00±0.00a

0.00±0.00a

0.00±0.00a

0.00±0.00a

2

26.03±3.69a

21.98±0.55a

25.42±3.25a

27.40±1.29a

23.15±3.34a

27.04±5.96a

3

39.36±3.07a

38.83±4.03a

38.47±4.48a

36.99±3.90a

30.79±1.62a

30.37±4.82a

4

39.36±3.07a

38.83±4.03a

38.47±4.48a

36.99±3.90a

30.79±1.62a

30.37±4.82a

1

0.00±0.00a

0.00±0.00a

0.00±0.00a

0.00±0.00a

0.00±0.00a

0.00±0.00a

2

78.46±1.54a

83.65±2.56a

82.42±2.77a

81.84±2.77a

81.48±1.75a

82.65±1.38a

3

82.31±2.31a

86.03±0.32a

84.98±0.36a

83.80±1.00a

83.56±2.63a

84.87±0.97a

4

82.31±2.31a

86.03±0.32a

84.98±0.36a

83.80±1.00a

83.56±2.63a

84.87±0.97a

1

0.00±0.00a

0.00±0.00a

0.00±0.00a

0.00±0.00a

0.00±0.00a

0.00±0.00a

2

29.88±2.02a

24.95±0.81a

27.00±1.66a

30.54±2.60a

26.91±1.31a

29.79±1.10a

3

46.58±2.34a

42.35±0.75a

44.79±1.22a

42.97±3.12a

40.14±4.67a

43.75±2.53a

4

46.58±2.34a

42.35±0.75a

44.79±1.22a

42.97±3.12a

40.14±4.67a

43.75±2.53a

1

77.76±0.48a

72.15±1.07a

76.61±2.85a

77.25±3.81a

78.57±2.03a

74.25±1.80a

2

77.76±0.48a

72.15±1.07a

76.61±2.85a

77.25±3.81a

78.57±2.03a

74.25±1.80a

3

77.76±0.48a

72.15±1.07a

76.61±2.85a

77.25±3.81a

78.57±2.03a

74.25±1.80a

4

77.76±0.48a

72.15±1.07a

76.61±2.85a

77.25±3.81a

78.57±2.03a

74.25±1.80a

1

0.00±0.00a

0.00±0.00a

0.00±0.00a

0.00±0.00a

0.00±0.00a

0.00±0.00a

2

35.05±2.51a

33.73±4.37a

32.46±6.42a

33.23±1.84a

35.45±5.79a

36.46±4.64a

3

66.92±4.59a

69.52±6.67a

64.52±1.40a

66.54±3.40a

67.58±2.58a

64.88±3.96a

4

90.46±0.26a

91.90±1.67a

90.32±0.16a

93.72±1.41a

92.12±2.88a

91.79±1.66a

1

5.25±2.72a

3.71±1.87a

5.59±3.06a

4.04±2.02a

3.98±2.03a

3.42±1.71a

2

42.61±3.47a

39.34±1.30a

41.15±2.17a

40.81±1.81a

39.73±3.23a

42.00±1.76a

3

46.51±2.06a

43.06±2.09a

42.91±0.47a

42.89±0.86a

45.79±2.99a

47.27±1.61a

4

46.51±2.06a

43.06±2.09a

42.91±0.47a

42.89±0.86a

45.79±2.99a

47.27±1.61a

1

4.74±2.47a

8.23±1.77a

5.79±3.22a

5.36±3.04a

4.78±2.63a

2.97±1.48a

2

39.38±2.05a

39.56±1.08a

44.58±2.92a

43.34±2.46a

42.91±1.30a

43.62±2.13a

3

43.30±1.88a

41.78±2.66a

48.66±3.55a

45.19±1.11a

44.67±1.83a

45.13±1.66a

4

43.30±1.88a

41.78±2.66a

48.66±3.55a

45.19±1.11a

44.67±1.83a

45.13±1.66a

1

0.00±0.00a

0.00±0.00a

0.00±0.00a

0.00±0.00a

0.00±0.00a

0.00±0.00a

Celosia argentea

Amaranthus retroflexus

Bidens pilosa

Echinochloa crusgalli

Dactyloctenium willd

Eleusine indica

31



Triticum aestivum

Page 32 of 51

2

41.35±0.83a

43.94±2.14a

43.47±1.93a

43.57±0.71a

42.60±0.75a

41.29±2.46a

3

41.35±0.83a

43.94±2.14a

43.47±1.93a

43.57±0.71a

42.60±0.75a

41.29±2.46a

4

41.35±0.83a

43.94±2.14a

43.47±1.93a

43.57±0.71a

42.60±0.75a

41.29±2.46a

1

78.57±3.91a

78.26±2.80a

75.93±1.85a

77.73±0.72a

77.78±2.78a

75.00±1.61a

2

82.27±2.35a

83.72±2.88a

79.63±1.85a

79.69±1.37a

79.86±2.50a

78.70±2.45a

3

82.27±2.35a

83.72±2.88a

79.63±1.85a

79.69±1.37a

79.86±2.50a

78.70±2.45a

4

82.27±2.35a

83.72±2.88a

79.63±1.85a

79.69±1.37a

79.86±2.50a

78.70±2.45a

1

89.10±2.89a

89.29±3.00a

89.93±2.66a

90.11±2.20a

88.85±2.79a

88.89±2.78a

2

89.10±2.89a

89.29±3.00a

89.93±2.66a

90.11±2.20a

88.85±2.79a

88.89±2.78a

3

89.10±2.89a

89.29±3.00a

89.93±2.66a

90.11±2.20a

88.85±2.79a

88.89±2.78a

4

89.10±2.89a

89.29±3.00a

89.93±2.66a

90.11±2.20a

88.85±2.79a

88.89±2.78a

Oryza sativa

702 703 704

The germination rates in the treatments did not change from 3 or 4 d to 7 d after the treatment. All data represent means ± SE. Values followed by different letters in the same line were significant differences (P < 0.05) according to Duncan’s multiple range test..

705 706 707 708 709 710 711 712 713 714 715 716 717

32


Page 33 of 51


718

Table 2 IC50 values of berberine on root length of some plants (7 d) r

IC50 (mg L−1) (95% CL)

y=3.9664+2.2271 x

0.9648

2.91(1.99-4.25)

Mikania micrantha

y=4.3693+1.6874x

0.9878

2.36 (1.64-3.40)

Celosia argentea

y=4.2976+1.8587x

0.9767

2.39 (1.85-3.07)


y=4.0338+1.6782x

0.9790

3.76 (2.68-5.28)

Bidens pilosa

y=4.3419+1.3676x

0.9799

3.03 (2.32-3.95)


y=3.7822+1.4726x

0.9958

6.71 (5.54-8.14)


y=3.6580+1.8249x

0.9954

5.44 (4.64-6.37)

y=3.2961+2.4906x

0.9782

4.83 (4.18-5.58)

Triticum aestivum

y=2.8594+2.1602x

0.9882

9.79 (9.12-10.52)

Oryza sativa

y=3.7256+1.5320x

0.9980

6.79（6.08-7.58）

Test plants Ageratum conyzoides

Dicot

Monocot

719 720

Eleusine indica

Regression equation

All treatments were replicated at least three times and repeated at least three times. IC50 was the median inhibition rates of the root lengths of tested weeds.

721 722 723 724 725 726 727 728 729 730 731 732 733 734 735 736 737 738 739 740 741 742 743 744 745 746

33



747

Table 3 IC50 values of berberine on fresh weight of some plants (7 d)

Dicot

Monocot

748 749

Page 34 of 51

Test plants

Regression equation

r

IC50 (mg L−1) (95% CL)

Ageratum conyzoides

y=3.9431+1.3904x

0.9637

5.76 (4.36-7.60)

Mikania micrantha

y=4.1526+1.0493x

0.9838

6.42 (5.15-8.01)

Celosia argentea

y=4.2069+1.0361x

0.9908

5.83 (4.99-6.80)


y=4.4723+0.7996x

0.9949

4.57 (2.97-7.04)

Bidens pilosa

y=4.0857+1.1297x

0.9958

6.45 (5.67-7.33)


y=3.4982+1.2234x

0.9807

16.89 (14.47-19.71)


y=3.7437+0.9854x

0.9544

18.83 (14.48-24.49)

Eleusine indica

y=3.0383+1.5839x

0.9798

17.32 (12.98-23.11)

Triticum aestivum

y=3.2755+1.1871x

0.9844

28.36 (26.63-30.20)

Oryza sativa

y=3.2593+1.1267x

0.9683

35.07（31.55-38.97）

All treatments were replicated at least three times and repeated at least three times. IC50 was the median inhibition rates of the fresh weight of tested weeds.

750 751 752 753 754 755 756 757 758 759 760 761 762 763 764 765 766 767 768 769 770 771 772 773 774 775

34


Page 35 of 51


776

Table 4 Change of biomass and thallus number of L. minor

Time(d) 3 5 7 9 11 14 777 778

Change of biomass (mg) Control 10 mg L-1 10.50±2.35a 31.20±2.09a 62.23±3.93a 89.23±1.44a 100.47±3.15a 181.90±22.42a

Change of thallus number Control 10 mg L-1

-2.70±0.59b -3.23±2.39b -5.30 ±3.31b -5.97±1.51b -13.5±1.19b -16.8±2.08b

21.33±0.67a 38.33±4.33a 57.67±7.22a 79.33±3.93a 110.00±2.65a 131.67±9.33a

0.00±0.00b 0.00±0.00b 0.00±0.00b 0.00±0.00b 0.00±0.00b 0.00±0.00b

All data represent means ± SE. Values followed by different letters in the same line were significant differences (P < 0.05) according to Student's t test.

779 780 781 782 783 784 785 786 787 788 789 790 791 792 793 794

35



Table 5 Change of chlorophyll content of L. minor (mg g-1)

795 Time (d)

796 797

Page 36 of 51

Chlorophyll a content (mg g-1)

Chlorophyll b content (mg g-1)

Total chlorophyll content (mg g-1)

Control

10 mg L-1

Control

10 mg L-1

Control

10 mg L-1

3

0.284±0.024a

0.120±0.019b

0.094±0.005a

0.047±0.007b

0.379±0.026a

0.166±0.026b

5

0.232±0.003a

0.030±0.002b

0.084±0.006a

0.013±0.002b

0.316±0.008a

0.043±0.004b

7

0.197±0.002a

0.000±0.000b

0.082±0.001a

0.000±0.000b

0.279±0.003a

0.000±0.000b

9

0.203±0.006a

0.000±0.000b

0.068±0.003a

0.000±0.000b

0.271±0.008a

0.000±0.000b

11

0.196±0.001a

0.000±0.000b

0.062±0.003a

0.000±0.000b

0.259±0.004a

0.000±0.000b

14

0.170±0.004a

0.000±0.000b

0.048±0.007a

0.001±0.001b

0.219±0.010a

0.007±0.004b

All data represent means ± SE. Values followed by different letters in the same line were significant differences (P < 0.05) according to Student's t test.

798 799 800 801 802 803 804 805 806 807 808 809 810 811 812 813 36


Page 37 of 51


814 815

816 817

-1

Table 6 Inhibition rate of chlorophyll content of L. minor treated with 10 mg L berberine

Time (d)

Chlorophyll a (%)

Chlorophyll b (%)

Total chlorophyll (%)

3 5 7 9

52.24±7.45c 87.03±0.88b 100.00±0.00a 100.00±0.00a

50.53±7.70c 84.56±1.76b 100.00±0.00a 100.00±0.00a

56.05±6.78c 86.37±1.09b 100.00±0.00a 100.00±0.00a

All data represent means ± SE. Values followed by different letters in the same row were significant differences (P < 0.05) according to Duncan’s multiple range test.

818 819 820 821 822 823 824 825 826 827 828 829 830 831 832 833 834 37



835

Table 7 Inhibition rate of plant fresh weight and primary root length of B. pilosa (%) (14 d)

Concentration (mg L-1) 2 4 8 16 32 836 837

Page 38 of 51

Inhibition rate of plant fresh weight (%) berberine glyphosate cinmethylin

Inhibition rate of primary root (%) berberine glyphosate cinmethylin

44.08±1.94b 53.15±1.77c 64.57±1.82c 80.91±0.91b 84.84±0.44b

46.49±1.83b 75.52±1.56b 84.84±1.81b 88.48±0.43b 94.64±0.36ab

69.49±1.36a 86.67±0.63a 90.52±1.57a 93.66±0.15a 95.56±0.25a

70.59±0.81a 79.94±1.18b 80.99±0.17b 82.62±1.00b 86.24±1.52b

79.57±0.71a 88.03±0.26a 93.28±1.13a 93.86±0.25a 95.40±0.08a

82.85±1.71a 86.25±0.24b 88.56±1.02ab 90.26±1.40b 93.63±0.48b

All data represent means ± SE. Values followed by different letters in the same line were significant differences (P < 0.05) according to Duncan’s multiple range test.

838 839 840 841 842 843 844 845 846 847 848 849 850 851 852 853 854 855

38


Page 39 of 51


856

Table 8 The LC50 of berberine on the B. rerio (96 h) Toxicity regression equation

Correlation coefficient

LC50 (mg L−1) (95% CL)

y=-10.0319 + 7.1522 x

0.9311

126.38 (115.99-145.78)

857

All treatments were replicated at least three times and repeated at least three times.

858 859 860 861 862 863 864 865 866 867 868 869 870 871 872 873 874 875 876 877 878 879 880 881 882 883 884 885 886 887 888 889 890 891 892 893 894 895 896

LC50 was the median lethal concentration.

39



897

Figure 1. Molecular structure of berberine

898 899 900 901 902 903 904 905 906 907 908 909 910 911 912 913 914 915 916

40


Page 40 of 51

Page 41 of 51


A

917 918

C

B

D

E

41



F

G

919 920

Figure 2. Inhibitory effect of 10 mg L-1 berberine on L. minor. (A): Overall view of L. minor; (B):

921

Change of biomass; (C): Change of thallus number; (D) Chlorophyll a content; (E): Chlorophyll b

922

content; (F): Total chlorophyll content; (G): Inhibition rate of chlorophyll content. Letters represent

923

values that differed significantly in the Student’s t test (B,C,D,E,F) and Duncan's multiple range test (G)

924

(P < 0.05). Shown are the averages of 30 plants ± SE (n = 3)

925 926 927 928

42


Page 42 of 51

Page 43 of 51


A

929 43



C

B

930 931

Figure 3. Inhibitory effect of berberine, glyphosate, cinmethylin, sulcotrione and 2,4-D at

932

different concentration on B. pilosa. (A): Overall view of B. pilosa; (B): Inhibition rate of fresh weight;

933

(C): Inhibition rate of primary root length. Letters represent values that differed significantly in the

934

Duncan's multiple range test (P < 0.05). Shown are the averages of 30 plants ± SE (n = 3)

935 936 937 938 939 940 941 942 943 944 945 946 947 948 949 950 951 952 953 954 955 956 957 958 959 960 44


Page 44 of 51

Page 45 of 51


18

a

B 18

1d

a

Inhibition rate of plants height (%)

Inhibition rate of roots elongation (%)

A

2d b

3d 5d

12

7d

c d a a a

6 a

a

a a

bb

b

2d

a a

3d

b

5d

12

7d c a ab

d

6

a ab ab

b c

0

bc c

c

0 root incubation

stem-coating

leaf-dipping

root incubation stem-coating

C

D 45

Inhibition rate of fresh plants weight (%)

Inhibition rate of fresh roots weight (%)

1d

1d

a

2d

a

3d 5d

b

30

7d

c a a

15

a

d

a

a a

a a

a

a

leaf-dipping

a

40

1d 2d 3d

b

30

5d c

20

7d

d a a

a

a

a a 10

a a e

a

b

0

0 root incubation

stem-coating

root incubation

leaf-dipping

stem-coating

leaf-dipping

961

Figure 4. Inhibitory effect of berberine with different application methods on (root incubation,

962

stem-coating and leaf-dipping ) B. pilosa. (A): Root inhibition rate; (B): Plant height inhibition rate;

963

(C): Fresh root weight inhibition rate; (D): Total fresh plant weight inhibition rate. Letters represent

964


965

of 30 plants ± SD (n = 3)

966 967 968 969 970

45


A

30

Concentration (µg g-1)


25

Page 46 of 51

leaf stem root

20 15 10 5 0 0.5d

1d

2d Time (d)

3d

5d

7d

971 40

C without B. pilosa

30

with B. pilosa

20 10 0 0.5d

1d

2d

3d

5d

Concentration (µg g-1)

Concentration (mg L-1)

B

7d

80

root

60

stem leaf

40 20 0 0.5d

1d

Time (d)

2d 3d Time (d)

5d

7d

972

Figure 5. Uptake of berberine by root incubation and leaf coating obtained from B. pilosa

973

seedlings. (A) Distribution of berberine in B. pilosa treated with 32 mg L−1 of berberine by root

974

incubation (B) The remaining amount of berberine in water solution with or without B. pilosa, B. pilosa

975

was treated as in (A); (C) Distribution of berberine with leaf coating (40 µL, 1000 mg L−1 ) Data are

976

means of 9 plants ± SE (n = 3).

977 978 979 980 981 982 983 984 985 986 46


Page 47 of 51


987

988

Figure 6. Representative fluorescence photographs of 6-d-old B. pilosa seedlings: The roots were

989

incubated in a solution containing 32 mg L−1 berberine. A1, B1, C1, D1, E1 and F1: roots; A2, B2, C2,

990

D2, E2 and F2: lower part of the stem; A3, B3, C3, D3, E3 and F3: upper stem; A4, B4, C4,D4, E4 and

991

F4: the first true leaf petiole; A5, B5, C5,D5 and E5: the second true leaf petiole; A (A1, A2, A3, A4

992

and A5), B (B1, B2, B3, B4 and B5), C (C1, C2, C3, C4 and C5), D (D1, D2, D3, D4 and D5), E (E1,

993

E2, E3, E4 and E5) and F (F1, F2, F3, F4 and F5) were control, 0.5, 1, 2, 3, 5 and 7 d, respectively. The

994

presence of fluorescent xenobiotics was indicated by arrowheads. P: pith. VB: vascular bundle.

995

47



Page 48 of 51

996 1200

a

800 b b 400 c d 0 0

4 8 16 Concentration (mg L−1)

32

PPO

c

c

4

a

0

6000

0

4

4000

8

16 32 b c

2000

e

d d

d

a b

c

0 0

4

8

16

32

1d 3d 5d 7d

a

b

40

20

a

SOD b

8 8000

POD

a

50

D

40 IUFA(%)

MDA concent (nmol•gFW-1)

a

a

Concentration (mg L−1)

60

C

12

10000

B Enzyme activity (U•gFW-1•min-1)

Root vigor (µg•g-1•h-1)

A

c

30

20

0 0

4 8 16 Concentration (mg L−1)

0

32

12.5

25

50

Concentration (mg

100 L−1)

997

Figure 7. Physiological effects of berberine on B. pilosa. A: Effects of berberine on root vigor of

998

B. pilosa; B:Effects of berberine on POD, PPO and SOD activity of B. pilosa root; C: Effects of

999

berberine on MDA of B. pilosa root; D: Effect of berberine on IUFA of B. pilosa. Letters represent

1000

values that differed significantly in the Duncan's multiple range test (P < 0.05). Data shown are the

1001

means ± SD (n = 3). Index of unsaturated fatty acid (IUFA): {[16 : 1 %×1] + [18 : 1 % ×1] + [18 :

1002

2 %×2] + [18 : 3 %×3]} ×100 %47.

1003 1004 1005 1006 1007 1008 1009 48


200

Page 49 of 51


1010 1011

1012 1013

A

B

C

D

E

F

Figure 8. SEM analysis of B. pilosa root surface under control (A and D) and treatment (B, E and C, F) with berberine (3.2 and 32 mg L−1)

1014 1015 1016 1017 1018 1019 1020 1021 1022 1023 1024 1025 1026 1027 1028 1029

49



1030

1031

Figure 9. TEM analysis of the ultrastructure changes in root cells treated with berberine. Root

1032

cells were treated with 3.2 and 32 mg L−1 berberine for 7 d, A, B, C: root cells treated with water only ,

1033

D, E, F: root cells treated with 3.2 mg L−1 berberine ;G, H, I: root cells treated with 32 mg L−1

1034

berberine.

50


Page 50 of 51

Page 51 of 51


For Table of Contents Only:

1035

51


Herbicidal Spectrum, Absorption and Transportation, and

Recommend Documents