Structure and Activity of the Camellia Oleifera Sapogenin Derivatives

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Functional Structure/Activity Relationships

Structure and Activity of the Camellia Oleifera Sapogenin Derivatives on Growth and Biofilm Inhibition of Staphylococcus aureus and Escherichia coli Chunfang Zhu, Meng Zhang, Qiaoling Tang, Qian Yang, Jing Li, Xuan He, and Yong Ye J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b03577 • Publication Date (Web): 30 Aug 2019 Downloaded from pubs.acs.org on August 30, 2019

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Journal of Agricultural and Food Chemistry

1

Structure and Activity of the Camellia Oleifera Sapogenin Derivatives on

2

Growth and Biofilm Inhibition of Staphylococcus aureus and Escherichia

3

coli

4

Chunfang Zhu1, Meng Zhang1, Qiaoling Tang1, Qian Yang1, Jing Li1*, Xuan

5

He2, Yong Ye1,2*

6

1 Department

7

Chemical Engineering, South China University of Technology, Guangzhou

8

510640, P R China

9

2

10

of Pharmaceutical Engineering, School of Chemistry and

Gannan Medical University Collaborative Innovation Center for Gannan

Oil-tea Camellia Industrial Development, Ganzhou 341000, P R China

11 12 13

Short title:

14

Antibacterial effects of the sapogenin derivatives

15 16

* Correspondence author:

17

Yong Ye, Jing Li

18

Department of Pharmaceutical Engineering, School of Chemistry and

19

Chemical Engineering, South China University of Technology, Guangzhou

20

510640, China

21

Tel: +86-20-87110234

22

Email: [email protected], [email protected]

1

ACS Paragon Plus Environment


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ABSTRACT: Sapogenin is the main block of Camellia oleifera saponin, which

24

was purified and structurally modified by C28 acylation reaction to synthesize

25

19 new derivatives. The growth and biofilm inhibition of Staphylococcus aureus

26

and Escherichia coli was measured to evaluate their antibacterial effects.

27

Three-dimensional quantitative structure-activity relationship (3D-QSAR)

28

assay indicated that the antibacterial activities were significantly enhanced

29

after the sapogenin was modified with aromatic ring or heterocyclic ring and

30

electron-withdrawing substituents at the meta or para position. Among them

31

the derivative of sapogenin with 2-mercapto-4-methyl -5-thiazolyl acetyl group

32

obviously destroyed bacterial biofilm and made bacteria lysis. 3D-QSAR

33

provides practical information for structural design of sapogenin derivatives

34

with strong antibacterial activity, and the Camellia oleifera sapogenin

35

derivative

36

tetrahydroxy- oleantel- 2-Ene- 23-aldehyde (S-16) is an effective candidate of

37

antibacterial agent for prevention of bacterial resistance against antibiotics.

28-O-(2-mercapto-4-methyl-5-thiazolyl)-

3β,16α,21β,22α-O-

38 39

KEYWORDS: sapogenin, derivative, Camellia oleifera, antibiofilm effect,

40

3D-QSAR assay, bacterial resistance

41 42 43 44

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45

INTRODUCTION

46

Antibiotics play important roles in the therapy of pathogenic microorganism

47

infection for a long time. However, due to abuse of antibiotics, the constant

48

increase of drug-resistant bacteria has become great threats to animals and

49

human being as well as severe challenges to therapy.1,2 It is meaningful to

50

develop alternative new antibacterial agents without resistance. Recent

51

researches focus on natural products because of their complex structure and

52

multiple targeting mechanisms,3-5 which can be exploited as antibiotic

53

substitutes with a lower propensity of bacterial resistance.

54

The saponin isolated from the seeds of Camellia oleifera Abel, has a wide

55

range of pharmacological activities, such as antibacterial, anti-inflammatory,

56

analgesic, insecticidal, anticancer, inhibition of alcohol absorption, lowering

57

blood lipids and so on,6,7 indicating its multi-targeting roles. As a by-product or

58

waste of oil manufacture, Camellia saponin has become an abundant, cheap

59

resource in China, and made it possible to be extracted as an antibiotic

60

substitute or drug candidate. Our previous research has found that the

61

Camellia saponin has less bacterial resistance.8 However, its effects and

62

qualities are unstable because of inconsistent structures even in the same

63

variety. In order to acquire the uniform structure of compounds we hydrolyzed

64

the saponin, and separated the main block called sapogenin from it.9 We have

65

also found that the sapogenin is the main active compound, but its activity

66

against Gram-negative bacteria is still low.8 It is possible to improve its activity

3



67

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by structural modification.

68

The sapogenin is a multi-hydroxyl structure (Figure 1), including secondary

69

hydroxyl group at C3, C16, C21, C22 and primary hydroxyl group C28 which is

70

active to be modified. The acylation reaction can improve the antimicrobial

71

activity of compounds.10 Therefore, we modified the structure of Camellia

72

oleifera sapogenin by C28 acylation reaction to synthesize a series of new

73

Camellia oleifera sapogenin derivatives, and tested their antibacterial and

74

antibiofilm activities on Staphylococcus aureus and Escherichia coli in order to

75

reveal the structure-activity relationship between sapogenin derivatives and

76

antibiofilm and find out useful antibacterial agents.

77

MATERIALS AND METHODS

78

Materials. The defatted seeds of Camellia oleifera were collected from The

79

Oil Factory in Meizhou of Guangdong province, China. The saponin was

80

isolated from the defatted seeds with methanol, and purified with macroporous

81

resin and successively hydrolyzed by acid and alkaline to obtain Camellia

82

oleifera

83

4-dimethylaminopyridine

84

3-ethylcarbodiinide hydrochloride (EDC·HCl) and Mueller-Hinton broth were

85

purchased from Shanghai Aladdin Biological Chemical Company (Shanghai,

86

China). Reagents for structural modification such as acetic anhydride,

87

chloroacetyl chloride, trifluoroacetic anhydride, maleic anhydride, benzoyl

88

chloride, m-chlorobenzyl chloride, m-fluorobenzyl chloride, m-bromobenzyl

sapogenin

in

our

research.9

previous

(DMAP),

Amoxicillin,

1-(3-dimethylaminopropyl)-

4


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89

chloride, m-methyl benzoyl chloride, 4-nitrobenzoyl chloride, 4-methoxy

90

benzoyl chloride, 2-thiophene formyl chloride, 2-furan formyl chloride, salicylic

91

acid,

92

2-mercapto-4-methyl-5-thiazole acetic acid,

93

chloride,

94

N,N-dimethylformamide (DMF), triethylamine (TEA) and other solvents were

95

purchased from Energy Chemical Company (Shanghai, China). All reagents

96

were of analytical without further purification.

5-bromosalicylic

acid,

tetrachloro

triphenylchloromethane

phthalic

anhydride,

p-acetylaminobenzenesulfonyl

(TrCl),

phthalic

anhydride,

97

Bacterial strains and culture. The control strains of Staphylococcus

98

aureus (ATCC 29213) and Escherichia coli (ATCC 25922) were purchased

99

from Guangdong Microbiology Culture Center (Guangdong, China). All

100

bacterial strains were activated, and cultured at 37ºC in MH broth. Drug

101

resistant E. coli and S. aureus strains were induced by amoxicillin according to

102

the reference.11

103

Synthesis of sapogenin derivatives. The synthetic route of Camellia

104

oleifera sapogenin derivatives was illustrated in Figure 2. Firstly, Camellia

105

oleifera sapogenin was dissolved in pyridine with its C28 primary hydroxyl

106

group protected by TrCl. Secondly, acetic acid anhydride was added to protect

107

C3, C6, C21 and C22 secondary hydroxyl groups in the condition of DMF as the

108

solvent, and TEA as the catalyst. Thirdly, formic acid was added to remove the

109

tritylmethyl group so that C28 hydroxyl group is exposed to react with a series

110

of acylation reagents by using DMAP as a catalyst.12 At last, the acetyl

5



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111

protecting groups of the secondary hydroxyl group were removed by acyl

112

chloride,13 and Camellia oleifera sapogenin derivatives were collected from

113

silica gel column chromatography with elution of petroleum ether/ethyl

114

acetate/acetic acid at volume ratio of 20/10/1. The detailed procedures for

115

intermediates 1a−3a and Camellia sapogenin derivatives are available in the

116

Supporting Information.

117

Structural Analysis. IR spectra were measured on VERTEX 70 FI-IR

118

spectrometer (Bruker Company, Germany) with KBr tablets from 4000 to 400

119

cm-1 with resolution 2 cm-1. Mass spectra were recorded on Bruker maXis

120

Mass spectrometer with ESI (Bruker Company, Germany) in m/z of cation

121

model scanning from 150 -1200 for 60 min. NMR spectra were determined on

122

400 MHz AM NMR (Bruker Company, Switzerland) in C2D6SO operating at

123

101 MHz for 13C NMR and 400 MHz for 1H NMR.

124

Structure-activity relationship. Comparative molecular force analysis

125

(CoMFA)

was

used

to

analyze

the

three-dimensional

126

structure-activity relationship (3D-QSAR) of Camellia oleifera sapogenin and

127

its derivatives.14 Twenty compounds were divided into a training set and a test

128

set, and molecular structures were optimized by Tripos force field and

129

Gasteiger-Huckel charge with the most active derivative as a template for

130

molecular superposition. The QSAR equation was obtained by partial least

131

square method (PLS) with a negative logarithm of 50% inhibition concentration

132

(pMIC50) as the dependent variable and molecular stereoscopic field energy or

6


quantitative

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133

electrostatic field energy as the independent variable. The optimum principal

134

component value and cross validation coefficient q2 were determined by

135

Leave-One-Out (LOO) method, and the correlation coefficient (r2), standard

136

deviation (s) and F value of 3D-QSAR model were obtained by

137

non-cross-validation method.

138

Bacteria growth inhibition and biofilm inhibition assay. The antibacterial

139

activity of Camellia oleifera sapogenin and its derivatives against S. aureus

140

and E. coli was tested using the microbroth dilution method.15 The bacteria

141

were diluted to 5×105 CFU/mL by sterile MH broth. The sapogenin derivatives

142

were dissolved respectively in DMSO to make 128 mg/mL solution, and diluted

143

by MH liquid medium (Contains 1% Tween 80) to make the series of final

144

concentrations at 2560, 1280, 640, 320, 160, 80, 40, 20, 10 and 5 μg/mL. A

145

similar method was used to dilute amoxicillin.

146

Sapogenin derivatives diluents (100 μL) and amoxicillin diluents (100 μL)

147

were sequentially added into the 96 well plates, which were inoculated with

148

100 μL tested bacteria (5×105 CFU/mL) in each well. Amoxicillin was used as a

149

positive control, and sterile MH broth, DMSO (1%, v/v) and Tween 80 (0.05%,

150

v/v) acted as negative controls. 96 well plates were incubated at 37°C for 24 h.

151

The optical density value at wavelength of 600 nm (OD600) were measured by

152

CYTATION5 micro-plate spectrometer (BioTeck Company, America) and

153

bacterial growth inhibition rate was calculated as follow. The minimum

154

inhibitory concentration on 50% bacterial strains was calculated as MIC50.

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Negative OD600  Drug OD600 100% Negative OD600

155

Bacterial growth inhibition rate(%) 

156

50 μL of negative control group suspensions were coated on Muller Hinton

157

agar evenly and cultivated at 37°C for 24 h. Bacterial colonies in the plates

158

were counted to calculate the number of bacteria in the corresponding wells.

159

Minimum

160

concentration of drug required to kill more than 99.99% of the tested strains

161

according to the CLSI standard.16,17

bactericidal

concentration

(MBC)

indicates

the

minimum

162

Bacterial biofilm experiments were carried out by crystal violet staining.18,19

163

After culture medium was removed from 96 well plates and washed 3 times

164

with PBS (0.1 M, pH=7.4), 0.1% crystal violet solution (100 μL) was added in

165

each well to dye the biofilm. 15 min later, the solution was aspirated. All wells

166

were washed 3 times with PBS (0.1 M, pH=7.4), and then 33% glacial acetic

167

acid solution (100 μL) was added to extract crystal violet from the biofilm. The

168

absorbance in 570 nm (OD570) was measured to calculate the biofilm inhibition

169

rate. The minimum concentration of drug required to inhibit biofilm formation by

170

50% of the tested strains was calculated as MBIC50. Negative OD570  Drug OD570  100% Negative OD570

171

Biofilm inhibition rate(%) 

172

Bacterial morphology observation. Transmission electron microscopy

173

was used to observe the morphology of S. aureus and E. coli. In short,

174

Bacteria were treated with 1× MBIC and 2 × MICs Camellia oleifera sapogenin

175

derivatives and washed with phosphate buffer saline (PBS) for three times.

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176

Then the samples were fixed with 2.5% glutaraldehyde for 12 h at 4°C and

177

rinsed twice with phosphate buffer saline (PBS) and further fixed with 1%

178

osmium tetroxide for 12 h, then dehydrated with a graded ethanol. The

179

obtained bacteria were embedded in a mixture of Spurr resin and acetone (v:

180

v=3:1) for 12 h, sectioned, stained with uranyl acetate and imaged under

181

JEM-1400 Plus transmission electron microscopy (Japan Electron Optics

182

Laboratory Co Ltd, Tokyo, Japan).

183

Biofilm integrity assay. The bacteria were respectively diluted to 2.3×108

184

CFU/mL, and added with Camellia oleifera sapogenin derivatives (1× MBIC, 2

185

× MICs, 4 × MICs,100 μL) or amoxicillin (1 mM,100 μL), and then 10 μL of

186

SYTOX GREEN dye (5 μM) was added and incubated at 37°C for 1 h. 10 μL of

187

each bacterial solution was transferred to a slide and observed under an IX83

188

inverted fluorescence microscope (Japan Olympus co., Ltd., Tokyo, Japan) at

189

420-485 nm of excitation wavelength. Fluorescent dots could be found if the

190

biofilm integrity is damaged.20

191

Interaction with Bacterial Mannitol -1-phosphate dehydrogenase.

192

Bacterial mannitol -1-phosphate dehydrogenase (M1PDH) is an important

193

factor in inducing bacterial resistance.21 The interaction between the

194

sapogenin or its derivatives and M1PDH was investigated by the protocol of

195

semi-flexible docking (CDOCKER) in Discovery Studio V3.0.8 Structural data of

196

M1PDH were downloaded from Protein Data Bank. After removing water

197

molecules and adding amino acid residues, hydrogen and force field to it, the 9



198

protein was defined as the receptor, the sapogenin and its derivatives

199

molecules were defined as ligands. CHARMm force field was applied to

200

ensure correct bond length, bond angle in a state of energy stability. Running

201

the program to obtain CDOCKER energy and CDOCKER interaction energy

202

which are two important parameters to analyze the affinity and action force

203

between the receptor and ligand.6

204

Activity of mannitol -1-phosphate dehydrogenase in bacterial biofilm.

205

The sapogenin, its derivatives and amoxicillin were diluted to final drug

206

concentration at 1000, 500, 250, 100, 50, 20, 10, 5 and 1 μM for 48 h bacteria

207

culture, the biofilm was homogenized in 500 μL of 50 mM phosphate buffer (pH

208

5.5), 10 μL of the supernatant was taken for enzyme activity determination.

209

The activity of bacterial mannitol -1-phosphate dehydrogenase (M1PDH) was

210

determined from the decrease in NADH by measuring the absorbance of

211

NADH at 340 nm.8 The reaction mixture contained sodium phosphate buffer

212

(200 mM, pH 5.5, 50 μL), NADH (2 mM, 50 μL), water (50 μL) and bacterial

213

biofilm extract (10 μL) . The mixture was maintained at 32°C for 2 min, and the

214

reaction was started by adding 40 μL of 1 M fructose and lasted for 5 min. The

215

absorbance at 340 nm was detected by UV-3010 spectrometer (Hitachi

216

Company, Japan).

217

Statistical Analysis. Data were expressed as mean ± standard deviation

218

( x  s ), and analyzed with SPSS17.0 software. Significant tests among the

10


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219

groups were based on one-way ANOVA and Student-Newman-Keuls (SNK)

220

test.

221

RESULTS AND DISCUSSION

222

Structure of the sapogenin derivatives. The purified sapogenin is

223

amorphous powder with a purity of 91.62% by HP1100 HPLC (Agilent

224

Company, USA) in the following operating conditions: column: Hypersil ODS

225

(250 × 4.6 mm, 5 μm); flow phase: methanol/water (80/20); injection volume:

226

10 μL; flow rate: 1 mL/min; temperature: 25°C; wavelength: 218 nm. IR spectra,

227

1H

228

formula C30H48O5, which is consistent with our former literature.9

NHR and

13C

NMR data showed that it had a sapogenin structure with

229

19 derivatives of sapogenin were synthesized with different substituent

230

groups in C28 position, and their yields are listed in Table 1. All products were

231

measured by IR, MS, 1H-NMR and 13C-NMR to confirm their structures.

232

Synthetic methods of Camellia sapogenin derivative S-(1-19) and the key

233

intermediates,1H NMR and

13C

234

sapogenin derivative S-(1-19) (C2D6SO as solvent) were shown in supporting

235

information file, which is available online.

NMR spectrum for sapogenin and Camellia

236

Antibacterial and anti-biofilm activity. With the solvents as a negative

237

control, the bacterial growth inhibition rate (MIC50), the minimum bactericidal

238

concentration (MBC) and the bacterial biofilm growth inhibition rate (MBIC50) of

239

the sapogenin and its derivatives (S-1 to S-19) against S. aureus and E. coli

240

were calculated to evaluate the effect of C28 hydroxyl modification of the

11



Page 12 of 39

241

sapogenin on antibacterial activity (Table 2). The results showed that

242

amoxicillin had no inhibition on bacterial growth and biofilm formation of S.

243

aureus and E. coli, suggesting bacterial resistant against amoxicillin.

244

Compared with the saponin and the sapogenin, antibacterial activity of the

245

sapogenin derivatives was significantly improved, especially the activity of

246

aromatic ring or heterocyclic ring and electron-withdrawing group modified

247

derivatives. It may be attributed to the structural change of the sapogenin and

248

its derivatives easily binding to the bacterial membranes, and taking effects

249

significantly.8,22

250

28-O-(2-mercapto-4-methyl-5-thiazolyl)-

251

oleantel- 2-Ene- 23-aldehyde (S-16) had the most powerful antibacterial and

252

antibiofilm activities, which is about three hundred times stronger than the

253

sapogenin. Its mechanism deserves further investigation.

Among

them

the

sapogenin

3β,16α,21β,22α-O-

derivative tetrahydroxy-

254

Structure-activity relationship. The parameters of the CoMFA model were

255

shown in Table 3. If the cross-validation coefficient q2>0.5 and the

256

non-cross-validation regression coefficient r2>0.8, then the obtained model has

257

reliable predictive ability.14 Therefore, a good predictive CoMFA model with the

258

cross-validation coefficient q2=0.556 and the non-cross-validation regression

259

coefficient r2=0.982 was established in this experiment. The model was used

260

to predict the biological activity of the training set and the test set compound

261

(Figure 3), the correlation between the predicted value and the experimental

262

value of the model reached 0.914 (p2202.42

373.38±7.23

719.40±8.41

>2202.42

768.44±17.89

S-3

459.80±7.59

>2130.85

377.81±2.41

553.52±7.92

>2130.85

504.74±8.99

S-4

476.15±6.06

>2123.60

439.73±10.04

650.85±8.10

>2123.60

662.96±2.65

S-5

131.23±2.18

525.62

124.42±6.99

322.77±4.24

2102.50

302.07±7.39

S-6

20.33±2.69

248.74

21.63±1.75

33.05±0.36

497.47

29.55±0.08

S-7

18.29±1.53

255.27

17.60±0.26

32.95±1.01

510.58

31.05±0.51

S-8

89.60±2.30

465.32

55.50±3.98

95.36±1.53

465.32

97.56±1.46

S-9

126.76±2.44

513.78

109.84±3.56

277.60±3.89

2055.14

269.09±3.37

S-10

53.07±2.02

244.72

41.30±1.79

70.69±1.79

489.45

72.36±1.18

S-11

89.79±0.97

500.92

78.69±2.22

142.64±2.19

500.92

131.83±1.26

S-12

11.36±0.68

65.06

8.24±0.15

17.71±1.24

130.12

16.34±1.08

S-13

120.82±3.27

2137.75

97.64±4.26

79.38±1.47

534.44

76.15±1.18

S-14

39.49±2.82

510.51

39.60±0.77

80.04±1.99

510.51

74.26±3.97

S-15

31.66±1.23

113.36

30.57±0.06

63.04±2.03

113.36

61.70±3.54

S-16

3.99±0.96

59.18

3.52±0.13

26.75±1.79

236.78

26.36±0.18

S-17

92.16±2.42

245.09

83.07±1.73

75.83±3.55

245.09

105.95±1.03

S-18

53.06±0.82

202.38

39.92±0.39

36.95±1.45

202.38

30.48±2.03

S-19

43.01±3.05

484.94

38.76±0.99

102.41±1.45

484.94

89.58±0.82

a

490

Notes:

MIC50 indicates the minimum inhibitory concentration of the drug

491

required to inhibit the growth of 50% of the tested strain. 25



492

b

493

99.99% of the tested strains.

494

c

495

formation by 50% of the tested strains. All data show significant differences

496

between treatments (p＜0.05) and are expressed as mean ± SD (n=3).

MBC indicates the minimum concentration of drug required to kill more than

MBIC50 indicates the minimum concentration of drug required to inhibit biofilm

497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513

26


Page 26 of 39

Page 27 of 39

514


Table 3. Statistical parameters of the CoMFA model Contribution（%）

515

Model

q2

r2

S

F

Steric

electrostatic

COMFA

0.556

0.982

0.14

47.527

69.6

30.4

CoMFA, comparative molecular force analysis.

516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 27



Page 28 of 39

534

Table 4. Binding energy of M1PDH with the sapogenin derivatives (mean ± SD,

535

n=5, kcal/moL) Compound

-Cdocker

-Cdocker

Compound

-Cdocker

-Cdocker

energy

interaction energy

energy

interaction energy

sapogenin

-83.61±5.58a

37.48±3.34b

S-10

-100.18±1.42a

52.02±2.20b

S-1

-89.62±3.54a

39.1±3.67b

S-11

-99.42±5.65a

61.24±4.63b

S-2

-81.55±2.89a

35.3±2.58b

S-12

-114.37±4.13a

43.33±2.86b

S-3

-89.7±1.52a

36.9±1.76b

S-13

-87.37±2.18a

52.38±1.87b

S-4

-96.02±4.25a

49.04±4.70b

S-14

-91.39±1.38a

63.75±6.65b

S-5

-109.95±4.93a

50.85±4.13b

S-15

-90.66±2.03a

56.96±2.11b

S-6

-97.41-±4.37a

57.02±4.72b

S-16

-109.87±4.54a

51.98±2.67b

S-7

-115.55±8.95a

44.46±4.62b

S-17

-110.11±4.3a

43.77±2.31b

S-8

-100.66±3.76a

56.08±2.09b

S-18

-96.67±1.56a

46.15±1.33b

S-9

-107.85±4.12a

48.44±1.06b

S-19

-104.46±7.98a

57.91±3.81b

Amoxicillin

31.62±0.86

49.71±2.07b

Celastrol

-218.76±9.42a

19.46±8.40

536

a

537

p

Structure and Activity of the Camellia Oleifera Sapogenin Derivatives

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