Characterization and Physicochemical Properties of Condensed

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Characterization and physico-chemical properties of condensed tannins from Acacia catechu Antoine Duval, and Luc Averous J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b05671 • Publication Date (Web): 08 Feb 2016 Downloaded from http://pubs.acs.org on February 13, 2016

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Journal of Agricultural and Food Chemistry

Characterization and physico-chemical properties of condensed tannins from Acacia catechu

Antoine Duval, Luc Avérous* Bioteam/ICPEES-ECPM, UMR 7515, Université de Strasbourg, 25 rue Becquerel, 67087 Strasbourg Cedex 2, France * Corresponding author: Prof. Luc Avérous, Phone: + 333 68852784, Fax: + 333 68852716, E-mail: [email protected]

1 ACS Paragon Plus Environment


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Abstract

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Condensed tannins from Acacia catechu were carefully studied to determine their chemical

3

structure and physico-chemical properties. The combined use of MALDI-TOF-MS and

4

NMR revealed that catechin and epicatechin are the predominant monomers. Most of the

5

compounds were dimers, as confirmed by size exclusion chromatography measurements. To

6

evaluate their potential as aromatic building block in polymer synthesis, special care was

7

given to the characterization and quantification of the different OH groups. A detailed

8

NMR analysis showed the predominance of catechin, with a catechin/epicatechin ratio of

9

4.2:1. Two distinct 1H NMR measurements confirmed the quantification. The thermal

10

properties were also determined: the tannins showed high temperature of degradation (ca.

11

190 °C) and glass transition temperature (ca. 140 °C), allowing for thermal processing or

12

chemical reactions at relatively high temperature. Acacia catechu tannins thus present

13

interesting features to be used as aromatic building block in polymer materials.

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Keywords

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tannins, Acacia catechu, 31P NMR, MALDI-TOF, 13C NMR, 1H NMR


13

31

C

P

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Introduction

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Tannins are polyphenolic plant metabolites, which are found in various proportions in all

18

vascular plants, as well as in some non-vascular plants, such as red-brown algae.1 Their

19

biological role is in most cases related to protection against infections or insects and

20

herbivories attacks.2 Thousands of compounds have been isolated and referred to as tannins

21

or plant polyphenols, making the definition of the term partially ambiguous.3 Three classes

22

of tannins are commonly defined: (i) the hydrolysable tannins, which are esters of gallic or

23

ellagic acids; (ii) phlorotannins, which are derivatives of phloroglucinol (1,3,5-

24

trihydroxybenzene); and (iii) condensed tannins.3

25

Condensed tannins, also called proanthocyanidins, are oligomers or polymers of flavan-3-ol

26

units.2 Several monomers are found in condensed tannins, differing from the OH

27

substitution of the A and B rings of the flavan-3-ol (Figure 1).4 Oligomers are formed by

28

oxidative coupling between flavanol monomers, mostly between positions 4 and 8 or 4 and

29

6,5 but can also involve phenyl ether bonds.2,4

30

As polyphenols, condensed tannins present a great potential for use in polymer science.1

31

Their multifunctional character makes them interesting precursors for the elaboration of

32

wood adhesives, and numerous studies have been published in that direction over the past 3

33

decades, with major contributions from Pizzi and co-workers.6–8 More recently, such tannins

34

have been widely studied for the elaboration of rigid9,10 and flexible foams,11 with

35

applications as insulating materials,12,13 adsorbent for wastewater treatment,14,15 or

36

precursor for carbon foams.16–18 The reactivity of tannin OH groups has also been exploited

37

to prepare different kinds of polymers, such as polyols by oxypropylation19 or

38

oxybutylation,20 or polyurethanes, either by conventional21,22 or non-isocyanate pathways.23 3 ACS Paragon Plus Environment


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Acacia catechu is a deciduous tree, mostly found in India and other countries in Southern

40

Asia, where it is designated as khair in Hindi or kachu in Malay.24 Acacia catechu heartwood

41

extracts are particularly rich in tannin and have long been used in traditional Indian and

42

Chinese medicines. They also enter in the composition of the betel quid (or paan), a

43

preparation commonly chewed in southern Asia for its stimulant and psychoactive effects.25

44

Potential health beneficial effects of Acacia catechu extracts have been largely studied, and

45

recently reviewed by Stohs and Bagchi.24 They include anti-inflammatory, tissue protectant,

46

antipyretic, antihyperglycemic, anticancer and analgesic activities.

47

However, only few studies reported on the chemical structure of Acacia catechu tannins.

48

Shen et al. studied methanol extracts of Acacia catechu heartwood and leaves by liquid

49

chromatography coupled to a mass spectrometry detector (LC-MS).26 Heartwood extracts

50

were mainly composed of catechin and epicatechin, with low levels of dimers. Leaves

51

extracts showed a higher content in esterified monomers, such as epicatechin-3-O-gallate

52

and epigallocatechin-3-O-gallate, together with the presence of flavonols (quercetin and

53

kaempferol) and caffeine. In an ethanol extract of heartwood, quercetin derivatives and

54

epicatechin were also detected, as well as gallic acid.27 In addition to catechin/epicatechin, Li

55

et al. also reported the presence in water extracts of Acacia catechu of rhamnetin, 4-

56

hydroxyphenylethanol and profisetidin, as well as 4 new uncommon phenolic compounds,

57

that they precisely assigned by NMR.28,29 Finally, hydrolysable tannins (gallic acid and ellagic

58

acid) were also extracted from Acacia catechu using ionic liquid and identified by LC-MS.30

59

All these studies were mainly focused on mono- or dimers extracted from Acacia catechu. To

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accurately analyze molecules of higher molar mass, Matrix-Assisted Laser Desorption-

61

Ionization (MALDI) coupled to time-of-flight (TOF) mass detector is a powerful technique. It


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has originally been applied to condensed tannins samples by Pasch et al.,31 and has since

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then been widely used to characterize the molecular architecture of various tannins, as

64

recently reviewed.32 It has for instance been applied to tannins from several Acacia species,

65

such as Acacia mearnsii,31,33 Acacia auriculiformis,34 Acacia mangium35 or Acacia confuse,36

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but to the best of our knowledge never to Acacia catechu.

67

In this study, we aimed at characterizing commercially available Acacia catechu tannin

68

oligomers, which have not been characterized in details until now, in order to evaluate their

69

potential for use in polymer applications. In addition to the common techniques used in

70

tannin characterization, MALDI-TOF mass spectrometry and

71

care was given to the determination and quantification of OH groups, using FTIR, 1H and 31P

72

NMR spectroscopies. The oligomeric nature was further revealed by size exclusion

73

chromatography (SEC). To evaluate what kind of chemical or thermal treatments could be

74

implemented without degrading the tannins, the thermal properties were carefully

75

evaluated by thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC),

76

and discussed in relation to their chemical structure.

77

Materials and Methods

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Materials

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Condensed tannins from Acacia catechu, Tan’Activ® CAT, were kindly supplied by Silva

80

Chimica (St. Michele Mondovi, Italy). As received, the tannins contained 5.8 wt% water. To

81

avoid any distortion of the experimental results, the samples were dried overnight at 70 °C

82

and stored in a desiccator prior to all the analysis carried out.

13


C NMR spectroscopy, special


83

Pyridine (sequencing grade, ≥ 99.5%) was purchased from Fisher Scientific, acetic anhydride

84

(ACS reagent, ≥ 97%) from Acros Organics, and methanol (laboratory reagent grade, ≥

85

99.6%), dichloromethane (puriss., ≥ 99%), 2,3,4,5,6-pentafluorobenzaldehyde (98%), 2-

86

chloro-4,4,5,5-tetramethyl-1,3,2-dioxaphospholane (95%), DMSO-d6 (99.9% D atoms) and

87

CDCl3 (99.8% D atoms) from Sigma-Aldrich. All chemicals were used as received without

88

further purification.

89

Acetylation

90

Acetylation was performed as previously reported on tannin samples.19,20 1 g of sample was

91

dissolved in 10 mL pyridine/acetic anhydride (1:1 v/v) and stirred at room temperature for

92

24h. Then, the sample was placed in an ice bath and 5 mL of methanol were added to

93

quench the reaction. The mixture was then transferred in a separating funnel with 40 mL

94

DCM, and successively washed with 2M HCl solution, saturated NaHCO3 solution and

95

deionized water. The organic phase was then dried over sodium sulfate and evaporated to

96

dryness in a rotary evaporator to yield the acetylated sample, which was finally dried

97

overnight in a vacuum oven at 40 °C.

98

MALDI-TOF mass spectrometry

99

The sample was dissolved in acetone/H2O (1:1 v/v). The matrix solution was freshly prepared

100

by dissolving to saturation super DHB (9:1 mixture of 2,5-dihydroxybenzoic acid and 2-

101

hydroxy-5-methoxybenzoic acid, Sigma Aldrich) in a H2O/CH3CN/HCOOH (50/50.1%)

102

solution. The sample and matrix solutions were then mixed in equal proportions, and 1 µL of

103

the resulting solution was deposited on the stainless steel plate.


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Mass spectra were acquired on a time-of-flight mass spectrometer (MALDI-ToF-ToF Autoflex

105

II ToF-ToF, Bruker Daltonics, Bremen, Germany) equipped with a nitrogen laser (λ = 337 nm).

106

An external multi-point calibration was carried out before each measurement using the

107

singly charged peaks of a standard peptide mixture (0.4 µM, in water acidified with 1%

108

HCOOH). Scan accumulation and data processing were performed with FlexAnalysis 3.0

109

software.

110

Fourier Transform Infrared Spectroscopy (FTIR)

111

FTIR spectrum was recorded in the attenuated total reflectance (ATR) mode on a Nicolet 380

112

FTIR spectrometer, in the range 400 – 4000 cm-1, as the average of 32 scans with 4 cm-1

113

resolution. The sample was directly deposited on the ATR crystal and carefully pressed to

114

ensure a good contact. The background was recorded with the empty ATR crystal in air.

115

Size Exclusion Chromatography (SEC)

116

Size exclusion chromatography (SEC) measurements were performed in chloroform (HPLC

117

grade) in a Shimadzu liquid chromatograph equipped with a LC-10AD isocratic pump, a DGU-

118

14A degasser, a SIL-10AD automated injector, a CTO-10A thermostated oven with a 5μ PLGel

119

Guard column, two PL-gel 5μ MIXED-C and a 5μ 100 Å 300mm-columns, and 2 online

120

detectors, a Shimadzu RID-10A refractive index detector and a Shimadzu SPD-M10A diode

121

array (UV) detector, respectively. Molar masses and dispersity were calculated from a

122

calibration with polystyrene standards. Acetylated tannin samples were dissolved in

123

chloroform and filtered through a 0.2 µm PTFE membrane. For all analyses the injection

124

volume was 50 µL, the flow rate 0.8 mL.min-1 and the oven temperature set at 25 °C.

125

1

H NMR



126

1

127

groups. An accurately weighed amount of sample (about 20 mg) was dissolved in 500 µL of

128

DMSO-d6. 100 µL of a standard solution of 2,3,4,5,6-pentafluorobenzaldehyde in DMSO-d6

129

were then added, and the spectrum was acquired on a Bruker 400 MHz spectrometer. The

130

quantification was based on the integration of the peaks from the methyl protons in

131

phenolic (2.4 – 2.1 ppm) and aliphatic acetyl groups (2.1 – 1.7 ppm).

132

In addition, 1H NMR was also conducted on the neat sample to specifically quantify the

133

phenolic OH groups as previously described on lignin samples.37 Prior to the analysis, the

134

tannin sample was dissolved in 0.004 M HCl solution and freeze-dried to allow a reliable

135

quantification of acidic and phenolic protons.37 An accurately weighed amount of sample

136

(about 20 mg) was then dissolved in 500 µL of DMSO-d6, 100 µL of a standard solution of

137

2,3,4,5,6-pentafluorobenzaldehyde in DMSO-d6 were then added, and 1H NMR spectrum

138

was recorded. Then, 150 µL D2O were added directly in the NMR tube, to allow the exchange

139

of labile protons with deuterium, and a second 1H NMR spectrum was recorded. The

140

amounts of COOH and phenolic OH groups were respectively determined as the differences

141

in the integrals in the 11 – 13 and 8 – 10 ppm regions between the spectra before and after

142

the addition of D2O.

143

31

144

31

145

protocols.38 An accurately weighed amount of sample (about 10 mg) was dissolved in 400 µL

146

of anhydrous CDCl3 / pyridine solution (1:1.6 v/v). 100 µL of a standard solution of

147

cholesterol (0.1 M in anhydrous CDCl3 / pyridine solution) containing Cr(III) acetylacetonate

148

as relaxation agent was then added. Finally, 100 µL of 2-chloro-4,4,5,5-tetramethyl-1,3,2-

H NMR spectra were measured on the acetylated samples to analyze and quantify the OH

P NMR P NMR was performed after phosphitylation of the samples, according to standard


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dioxaphospholane (Cl-TMDP, 95%, Sigma-Aldrich) were added and the mixture was stirred at

150

room temperature for 2h. The mixture was then transferred into 5 mm NMR tubes and the

151

spectra were measured on a Bruker 400 MHz spectrophotometer (128 scans at 20 °C). All

152

chemical shifts reported are relative to the reaction product of water with Cl-TMDP, which

153

gives a sharp signal in pyridine/CDCl3 at 132.2 ppm. Peaks assignations and quantitative

154

analysis were performed based on previous reports.39,40

155

13

156

About 100 mg of sample were dissolved in 650 µL of DMSO-d6. 2-3 mg Cr(III) acetylacetonate

157

were added to the mixture to reduce the relaxation delay.41,42 The spectrum was acquired on

158

a Bruker 400 MHz spectrophotometer, with a 1.8 s acquisition time and a 2 s delay. 15 000

159

scans were collected. The peak assignation was based on previous literature reports.43,44

160

Differential Scanning Calorimetry (DSC)

161

DSC thermograms were recorded on a TA DSC Q200 calorimeter (TA Instruments). The

162

samples were first heated from room temperature to 150 °C, and maintained at this

163

temperature for 10 min in order to erase thermal history. They were then cooled to -60 °C

164

and finally heated up to 180 °C at a 10 °C.min-1 heating rate. The glass transition

165

temperature (Tg) was measured as the midpoint of the change in slope on the second

166

heating ramp. All experiments were run on triplicates, and results are given as average and

167

standard deviations.

168

Thermogravimetric analysis (TGA)

C NMR



169

TGA was measured on a TGA Q5000 apparatus (TA Instruments), under helium or air. The

170

samples were heated from room temperature to 700 °C at a 20 °C.min-1 heating rate. All

171

experiments were run on duplicates.


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Results and Discussion

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Chemical and structural characterization by MALDI-TOF MS and 13C NMR

174

MALDI-TOF mass spectrometry allows the characterization of high molar mass molecules

175

without fragmentation, and has thus been widely used for the characterization of various

176

condensed tannins.32 The spectrum of Acacia catechu tannins is displayed on Figure 2a. The

177

most prominent peaks are located in the region 600 – 700 Da (Figure 2c), which corresponds

178

to dimers. Other important peaks are located between 280 and 500 Da (Figure 2b), and

179

correspond to the monomers present in Acacia catechu tannins. Two other groups of peaks

180

are assigned to trimers (840 – 940 Da) and tetramers (1130 – 1210 Da), but oligomers of

181

higher degree of polymerization (DP) are not detected. This indicates that Acacia catechu

182

tannins possess a quite low DP compared to other wood tannins: maximum DPs of 8 to 12

183

were for instance measured on tannins from other Acacia species.31,33–36

184

Figure 2b shows the detail of the monomer region of the spectrum. 3 main peaks are located

185

between 295.2 and 328.7 Da. They can be assigned to the 4 commonly reported monomers

186

of condensed tannins (Figure 3): profisetidin (B, [M + Na]+ = 297.3 Da), catechin and/or

187

prorobinetinidin (C, C’, [M + Na]+ = 313.3 Da) and gallocatechin (D, [M + Na]+ = 329.3 Da).

188

MALDI-TOF-MS alone does not allow distinguishing between structures of same molar mass.

189

The 13C NMR spectrum presented on Figure 4 can provide the complementary information.

190

The signal of C1’, showed on the inset on Figure 4, allow distinguishing between catechol

191

and pyrogallol B rings.44 The main peak at 131.0 ppm corresponds to catechols, whereas the

192

small one at 133.5 ppm is attributed to pyrogallols. Thus, Acacia catechu tannins contain

193

almost exclusively catechol B rings. Signals assigned to the heterocyclic C ring also appear



194

clearly, showing that the isolation process did not induce ring opening reactions, as reported

195

for instance when sulfite processes are involved.1

196

The peak at 313.3 Da on the MALDI spectrum can then be attributed to catechin (C) rather

197

than to prorobinetinidin (C’). The presence of 3,5,7,4’-tetrahydroxyflavan (B’), reported for

198

example in Pinus brutia bark tannins,45 together with or in place of profisetidin (B), cannot

199

be determined with this technique. In addition, a peak is also detected at lower m/z and

200

could be assigned to a trihydroxy flavan ([M + Na]+ = 281.3 Da), as previously reported for

201

Pinus brutia.45 It could originate from the loss of two OH groups from catechin during the

202

ionization process. However, the intensities of all these peaks are low, indicating that they

203

are only present in trace amounts.

204

Three groups of peaks located between 390 and 430 Da show a higher intensity. Their molar

205

masses correspond to derivatives of the aforementioned monomers, which would be

206

esterified with dihydroxybenzoic acid. The presence of flavanols esterified at C3 with p-

207

hydroxybenzoic acid or 3,4-dihydroxybenzoic acids have been mentioned in recent work on

208

Pinus brutia bark tannins.45 However, neither the 13C NMR (Figure 4) nor the FTIR spectrum

209

(Figure 5) display any signal characteristic of esters. It thus seems that esters do not exist in

210

the native state, and would only be formed during the ionization process. Indeed, the

211

encountered molar masses correspond to esters of the flavanols units and molecules from

212

the matrix, namely 2,5-dihydroxybenzoic acid (DHB, Figure 6). It is interesting to note that

213

they appear on the [M + H]+ form, rather than on the [M + Na]+ form (Table 1). The

214

coexistence of multiple ion forms has previously been reported for similar materials.45,46


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215

The dimers, trimers and tetramers have then been assigned as combinations of the 4

216

monomers reported, with or without esters. All assignations are given in Table 1. The

217

theoretical masses are calculated according to Equation 1:

218

= ∑ − 2 × − 1 + + (1)

219

where Mmonomers is the molar mass of individual monomers, DP is the degree of

220

polymerization, Mester is the molar mass increment caused by esterification (Figure 6) and

221

MNa is the mass of sodium atom.

222

In some cases, the theoretical mass is found to be greater than the experimental mass. This

223

phenomenon, usually reported in the study of tannins by MALDI-TOF, is due to the

224

aromatization of the heterocycle under irradiation (Figure 7).45 The aromatization causes the

225

loss of 3 H atoms, and thus a decrease of 3 Da. In oligomers, the aromatization of several

226

heterocycles can cause some small differences in molar masses, up to 3 times the DP.

227

From Table 1, it can clearly be seen that most of the relevant peaks originates from

228

oligomers containing catechin/epicatechin, which thus seems to be the major repeat unit in

229

Acacia catechu tannins.

230

Molar mass distribution

231

The molar mass distribution of Acacia catechu tannins, as measured by SEC in chloroform on

232

acetylated samples, is given on Figure 8. The main peak on the distribution is located around

233

600 g.mol-1. This value corresponds to the approximate molar mass of dimers, and thus

234

confirms their predominance in Acacia catechu tannins, in good agreement with MALDI-TOF

235

data. Other peaks are discernible at about 285 g.mol-1 (monomers), 1110 g.mol-1 (trimers)



236

and 1580 g.mol-1 (tetramers). In addition, tailing up to about 7000 g.mol-1 suggests the

237

presence of oligomers of higher DP, which were however not visible on MALDI-TOF spectra.

238

Based on the calibration with polystyrene standards, the number- and mass-average molar

239

masses of Acacia catechu tannins are respectively Mn = 914 g.mol-1 and Mw = 1407 g.mol-1,

240

which corresponds to a dispersity Ð of 1.54.


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Structural characterization by 31P NMR

242

31

243

technique largely used for the characterization of lignin samples.38,47 It was recently adapted

244

to the study of tannins by Melone et al.39,40 and since then used to study various tannin

245

samples.19,20 A great advantage of this technique is the ability to distinguish and quantify

246

phenolic groups depending on their ortho-substitution pattern. In condensed tannins, it is

247

thus possible to separate non-substituted phenols, mono-substituted phenols, catechols or

248

di-substituted phenols. 31P NMR spectrum of Acacia catechu tannins is displayed on Figure 8,

249

together with the assignation of the main peaks, assuming that catechin is the most relevant

250

unit.

251

In the aliphatic OH region, a strong peak is observed at 145.3 ppm. It corresponds to the

252

aliphatic OH groups attached to the heterocycle with a 3S configuration, as in (+)-catechin

253

(Figure 9). The peak located at 145.9 ppm is attributed to a 3R configuration, as in (-)-

254

epicatechin.40 Based on the integration of the corresponding peaks, the catechin/epicatechin

255

ratio was estimated to 4.2:1. For comparison, this ratio was also estimated from the 13C NMR

256

spectrum, by comparing the integrals of C2 in trans (81.4 ppm) and cis units (78.5 ppm),

257

shown on the inset on Figure 4.43 A 3.7:1 ratio was obtained, confirming the order of

258

magnitude found with

259

stereoisomerism in tannins, as compared to 13C or 2D NMR measurements requiring longer

260

acquisition times.48

261

Some signal is also detected in the 149 – 146 ppm region, corresponding to other aliphatic

262

OH groups.38 In oligomers, a linkage at the C4 position, commonly observed in condensed

263

tannins, might cause a change in chemical shift. The presence of impurities, such as mono-

P NMR of samples phosphorylated with 2-chloro-4,4,5,5-tetramethyldioxaphospholane is a

31

P NMR.

31

P NMR thus appears as a quick way to quantify the



264

or oligomeric sugars or lignin fragments could also explain the additional aliphatic OH groups

265

which are detected.

266

No signal is observed in the o-disubstituted phenolic OH region (141.5 – 143.5 ppm),

267

confirming that the B rings in Acacia catechu tannins are almost exclusively catechols, and

268

not pyrogallols. It also indicates that gallic acid or its ester derivatives, such as catechin-3-O-

269

gallate, are absent, in good agreement with the results of MALDI-TOF-MS. In addition, a

270

linkage at position 6 would also cause the OH group at position 5 to be o-disubstituted. The

271

absence of signal in this region thus indicates that the linkages between catechin monomer

272

units do not involve C4-C6 bonds in Acacia catechu tannins.

273

In the catecholic OH region (138.3 – 140.5 ppm), a main peak is obtained at 138.9 ppm. In

274

the o-substituted OH region (137.8 – 138.3 ppm), the main peak at 138.1 ppm can be

275

assigned to the OH group at position 5.39 A linkage in position 8 would cause the OH group at

276

position 7 to be o-substituted, and could be the origin of the second peak observed at 137.9

277

ppm. Finally, the peak at 137.7 ppm is related to the o-unsubstituted OH group at position 7

278

in terminal units.

279

In flavonols, the presence of a carbonyl group in position 4 results in an upfield signal for the

280

phenolic OH at position 5, for which a chemical shift of 136.5 ppm was observed.39 The

281

absence of signal in this region reveals that flavonols, such as quercetin, kaempferol or

282

rhametin, previously detected in other studies,26,29 are not present in significant amounts in

283

the studied Acacia catechu tannins.

284

The results of the quantification of the different types of OH groups are listed in Table 2. The

285

experimental ratios are also compared with the theoretical ones for catechin/epicatechin

286

monomer. A slightly higher value than expected is measured for catechols (2.36 ± 0.05), but 16 ACS Paragon Plus Environment

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the ratio of aliphatic to total phenolic OH (1 to 4.16 ± 0.09) is consistent with catechin as

288

main monomer unit. The interunit linkages, which modify the ortho-substitution of the

289

corresponding phenolic OH groups, as well as signals overlap in the 136.5 – 140.5 ppm can

290

cause the slight deviation with the theoretical ratios of the monomer.

291

Identification and quantification of OH groups by NMR

292

When tannins aim at being chemically modified, for instance to be used as building blocks in

293

polymers, its numerous OH groups appear as a very attractive platform for chemical

294

reactions.1 A good knowledge of the nature and quantity of these groups is thus an

295

important prerequisite for the development of tannin-based polymer materials. In addition

296

to

297

into the OH composition of Acacia catechu tannins. All experiments were run in triplicate, in

298

order to evaluate the reproducibility of each method.

299

First, 1H NMR spectra were measured on neat samples dissolved in DMSO-d6. Right after the

300

measurement, D2O is added in the tube and another spectrum is acquired (Figure 10a). In

301

presence of D2O, all the labile protons, such as phenolic OH and COOH, are exchanged with

302

deuterium, causing the decrease of the NMR signal in the 8 – 10 and 10 – 13 ppm regions,

303

respectively.37 The difference in NMR signals before and after the addition of D2O thus gives

304

access to their quantification. In the carboxyl region (11 – 13 ppm), no signal is detected

305

before the addition of D2O (Figure 10a), confirming the absence of free acids, such as gallic

306

acid, as already noticed by MALDI-TOF-MS and

307

ppm), the addition of D2O causes a strong decrease in the NMR signal (inset on Figure 10a).

308

The content of Acacia catechu tannins in phenolic OH groups was thus found to be 10.47 ±

31

P NMR, two distinct 1H NMR measurements were performed in order to gain insights

31

P NMR. In the phenolic OH region (8 – 10



309

0.55 mmol.g-1. Three small peaks located at 7.9, 8.4 and 8.7 ppm remain after the addition of

310

D2O. They reveal the presence of trace amounts of aldehydes.37

311

1

312

the –CH3 peaks of the acetyl groups give the aliphatic (1.7 – 2.1 ppm) and phenolic OH

313

groups (2.1 – 2.4 ppm) contents. The corresponding values are 3.46 ± 0.04 and 8.05 ± 0.09

314

mmol.g-1 for aliphatic and phenolic OH, respectively.

315

Table 3 summarizes the results obtained from all the NMR measurements. The

316

reproducibility of 31P NMR and 1H NMR determination on acetylated samples is very good,

317

but it is poorer for 1H NMR on neat samples, with a relative standard deviation slightly

318

higher than 5%.

319

The absolute values determined with the different techniques appear however relatively far

320

from each other (Table 3). Similar discrepancies between quantification techniques have

321

already been evidenced several times on lignins.49–51 The phenolic OH content appears

322

significantly lower when measured by 1H NMR on acetylated samples rather than with the

323

two other techniques. This underestimation can be caused by an incomplete acetylation,

324

especially in highly substituted aromatic rings, where steric hindrance can alter the

325

reaction.50,51 However, the FTIR spectrum of the acetylated sample showed the total

326

depletion of the OH band around 3400 cm-1 (data not shown). A small loss of material during

327

the acetylation steps could also be involved. The 1H NMR determination on neat samples

328

gives on the contrary a phenolic OH content significantly higher than the other techniques

329

(Table 3), in agreement with results previously reported for lignin samples.37 Finally, the

330

quantification of phenolic OH groups obtained with 31P NMR corresponds to the average of

331

the three measurements techniques. Additional experiments on a broader range of tannin

H NMR spectra were also measured on acetylated samples (Figure 10b). The integrations of


Page 18 of 46

Page 19 of 46


332

samples with different macromolecular structures should later be conducted in order to

333

better understand the differences between the quantification techniques.

334

Thermal properties

335

The thermal properties of Acacia catechu tannins were determined. TGA was performed

336

under helium and air, in order to study the thermal degradation under both inert and

337

oxidizing atmospheres. The thermograms are depicted on Figure 11a, together with the

338

curves of the derivative weight loss (DTG). A first mass loss below 100 °C is caused by the

339

loss of water which was not removed by the preliminary drying step. The tannins are

340

thermally stable up to about 190 °C, which is an evidence of their good purity, since the

341

presence of residual carbohydrates has been shown to strongly decrease the onset of

342

thermal degradation.52,53 The thermal degradation occurs then over a relatively wide

343

temperature range, with a first degradation peak located at 294 ± 3 °C, higher than

344

previously reported for sumac,53 quebracho or pine bark tannins.54 Under helium, the

345

sample continues to degrade slowly at higher temperature, to finally show a residual mass of

346

36 ± 3% at 700 °C. The residual mass is likely to result from heat-induced polymerization,

347

which limits the sample’s pyrolysis.55 Under air, combustion occurs between 400 and 500 °C,

348

with a strong maximum in the degradation rate at 486 ± 1 °C, corresponding to the release

349

of the oxidized residues (CO2, CO, H2O).53 The ash content of the tannin sample was found to

350

be 0.15%.

351

DSC was measured on neat and acetylated Acacia catechu tannin samples. The thermograms

352

of the second heating run are displayed on Figure 11b. Only one thermal event,

353

corresponding to the glass transition (Tg), is observed for the analyzed thermal domain. Tg

354

was found to be 140 ± 1 °C for the unacetylated sample. Considering the low molar mass of 19 ACS Paragon Plus Environment


Page 20 of 46

355

Acacia catechu tannins, the Tg appears quite high, as compared for instance with lignins of

356

similar molar mass (16.3 °C for lignosulfonates of Mn = 1030 g.mol-1 or 70 °C for Kraft lignin

357

of Mn = 1200 g.mol-1).56,57 In lignins, the presence of ether bonds contributes to reduce the

358

Tg,58 whereas tannin monomer units are only linked by C-C bonds. This leads to a reduced

359

chain mobility and thus a higher Tg for tannins than for lignins. Derivatization of the OH

360

groups by acetylation causes a strong decrease of Tg, which was measured at 70 ± 1 °C

361

(Figure 11b). This originates from the blocking of the subsequent H bonds, as well as from

362

the concomitant increase in free volume caused by the introduction of the acetyl groups.

363

Similar reduction in Tg upon derivatization of OH groups has been similarly reported on

364

tannin or lignin samples modified by e.g. methylation59 or oxypropylation.19,59,60

365

In conclusion, the combined use of MALDI-TOF mass spectrometry,

366

spectroscopies revealed that Acacia catechu tannins are mostly composed of catechin and

367

epicatechin flavanol units, in a 4.2:1 ratio. They thus differ from other Acacia species and

368

from common commercial wood tannins, such as quebracho or mimosa (Table 4). They are

369

structurally closer to pine bark tannins, but possess a lower DP, as revealed by both MALDI-

370

TOF and SEC measurements (Table 4). The lower DP can however be seen as an advantage

371

for a use as aromatic building block, for the synthesis of e.g. bio-based polymer materials. In

372

addition, the good thermal stability of Acacia catechu tannins is compatible with many

373

chemical reactions or thermal processes, and confirms their potential for the elaboration of

374

innovative aromatic macromolecular architectures, for instance.


31

P and 1H NMR

Page 21 of 46


375

Acknowledgments

376

Silvateam (Italy), and more particularly Dr. Samuele Giovando, is gratefully acknowledged for

377

kindly supplying Acacia catechu tannin samples. Pr. Antonio Pizzi (Université de Lorraine,

378

France) is thanked for previous and helpful exchanges. Chheng Ngov (ICPEES, Université de

379

Strasbourg) is thanked for her technical support.

380



381

References

382

(1) Arbenz, A.; Avérous, L. Chemical modification of tannins to elaborate aromatic

383 384 385 386

biobased macromolecular architectures. Green Chem. 2015, 17, 2626–2646. (2) Khanbabaee, K.; van Ree, T. Tannins: classification and definition. Nat. Prod. Rep. 2001, 18, 641–649. (3) Quideau, S.; Deffieux, D.; Douat-Casassus, C.; Pouységu, L. Plant polyphenols: chemical

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(4) Aron, P. M.; Kennedy, J. A. Flavan-3-ols: nature, occurrence and biological activity. Mol. Nutr. Food Res. 2008, 52, 79–104. (5) Schofield, P.; Mbugua, D. M.; Pell, A. N. Analysis of condensed tannins: a review. Anim. Feed Sci. Technol. 2001, 91, 21–40.

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(7) Pizzi, A. Recent developments in eco-efficient bio-based adhesives for wood bonding:

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opportunities and issues. J. Adhes. Sci. Technol. 2006, 20, 829–846. (8) Pizzi, A. Chapter 8 - Tannins: major sources, properties and applications. In Monomers,

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polymers and composites from renewable resources; Gandini, A., Belgacem, M. N., Eds.;

398

Elsevier: Amsterdam, 2008; pp 179–199.

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quantitative lignin characterization by NMR spectroscopy. J. Agric. Food Chem. 2004,

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52, 1850–1860.

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Drake, C.; Grabber, J. H. 1H–13C HSQC NMR spectroscopy for estimating

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in lignins evaluation of 1H-, 13C-, 31P-NMR, FTIR and wet chemical methods.

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Characterization of sumac (Rhus tripartitum) root barks tannin for a potential use in

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wood adhesives formulation. Wood Sci. Technol. 2014, 49, 205–221.

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(54) Luo, C.; Grigsby, W.; Edmonds, N.; Easteal, A.; Al-Hakkak, J. Synthesis, characterization,

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and thermal behaviors of tannin stearates prepared from quebracho and pine bark

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extracts. J. Appl. Polym. Sci. 2010, 117, 352–360.

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polyphenols (vegetable tannins): preliminary results. Org. Mass Spectrom. 1992, 27,

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ultrafiltration and ethanol solubility to obtain a set of fractions with distinct properties.

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mechanical properties of kraft lignin by ultrafiltration. J. Appl. Polym. Sci. 2014, 131,

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9505–9515.

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(58) Baumberger, S.; Dole, P.; Lapierre, C. Using transgenic poplars to elucidate the

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relationship between the structure and the thermal properties of lignins. J. Agric. Food

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Chem. 2002, 50, 2450–2453.

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(59) Cui, C.; Sadeghifar, H.; Sen, S.; Argyropoulos, D. S. Toward thermoplastic lignin

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polymers; Part II: Thermal & polymer characteristics of Kraft lignin & derivatives.

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Bioresources 2013, 8, 864–886.

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Characterization of chain-extended hydroxypropyl lignins. J. Wood Chem. Technol.

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1988, 8, 341–359.

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(61) Jerez, M.; Sineiro, J.; Guitián, E.; Núñez, M. J. Identification of polymeric procyanidins

535

from pine bark by mass spectrometry. Rapid Commun. Mass Spectrom. 2009, 23, 4013–

536

4018.

537

(62) Navarrete, P.; Pizzi, A.; Pasch, H.; Rode, K.; Delmotte, L. MALDI-TOF and 13C NMR

538

characterization of maritime pine industrial tannin extract. Ind. Crops Prod. 2010, 32,

539

105–110.

540


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541

Figure captions

542

Figure 1. General chemical structure and nomenclature of condensed tannin monomers, based on

543

flavan-3-ol units.

544

Figure 2. MALDI-TOF MS spectrum of Acacia catechu tannins: full spectrum (a) and details of the

545

monomer (b), dimer (c) and tri- and tetramer (d) regions.

546

Figure 3. Main monomers structures detected by MALDI-TOF-MS for Acacia catechu tannins.

547

Figure 4. 13C NMR spectrum of Acacia catechu tannins. The insets show the C1’ and C2 regions

548

respectively. The peaks assignation is based on previous literature reports.43

549

Figure 5. FTIR spectrum of Acacia catechu tannins.

550

Figure 6. Esterification of catechin with molecules from the matrix during MALDI-TOF experiments.

551

The formed product has a molar mass increased by 136 as compared to the initial monomer.

552

Figure 7. Aromatization of the heterocycle (C ring) of catechin, causing a 3 Da difference between the

553

measured experimental mass and the theoretical one.

554

Figure 8. Molar mass distribution of acetylated Acacia catechu tannins measured by SEC in

555

chloroform.

556

Figure 9. 31P NMR spectrum of Acacia catechu tannins. The peaks assignation is based on previous

557

literature reports.39,40 IS = Internal standard (cholesterol).

558

Figure 10. 1H NMR spectra of Acacia catechu tannins: (a) neat samples in DMSO-d6 before (black) and

559

after addition of D2O (red), the inset shows the detail of the phenolic protons region, (b) acetylated

560

sample in DMSO, detail of the acetyl protons region.



561

Figure 11. Thermal analysis of Acacia catechu tannins: (a) TGA thermograms, showing the weight loss

562

(solid lines) and derivative weight loss (dashed lines) vs the temperature under helium or air; (b) DSC

563

thermograms of neat and acetylated samples (second heating run).


Page 30 of 46

Page 31 of 46


Tables Table 1. Assignation of the main peaks of the MALDI-TOF spectrum of Acacia catechu tannins.

Monomers

Dimers

Experimental peak (Da) 282.5 295.2 312.3 328.7 395.2 411.2 427.3

Theoretical peak (Da) 281.3 297.3 313.3 329.3 394.3 410.3 426.3

563.3

569.6

573.3

578.6

2

621.4

619.6

1

627.4

737.6

2

1

1

643.4

737.6

2

1

1

863.4

866.9

3

891.4

889.9

3

913.5

1025.9

3

1

1

933.5

1025.9

3

1

1

1135.5 1146.5

1146.2

1171.5

1178.2

A 258.3

B 274.3

C 290.3

D 306.3

Estera (+ 136)

1 1 1 1 1 1

1 1

Comments

1 1 1 1

1

1

Na 23

2

1

1

[M + H]+ form [M + H]+ form [M + H]+ form Aromatization of C rings (- 6 Da) Aromatization of C rings (- 6 Da)

1 Loss of catechol ring (- 110 Da) Loss of phenol ring (- 94 Da)

845.4

Trimers

Tetramers a

2

Aromatization of C rings (- 6 Da) 1

2

1

4

1

1203.6 1203.2 1 Esterification with 2,5-dihydroxybenzoic acid (Figure 6)

3


Loss of catechol ring (- 110 Da) Loss of phenol ring (- 94 Da)

Aromatization of C rings (- 6 Da) [M + H]+ form


Page 32 of 46

Table 2. Quantification of the different phenolic OH groups by 31P NMR (averages and standard deviations of three distinct measurements)

Functional group

Integration range (ppm)a

Amount (mmol.g-1)

Experimetal ratiob

Aliphatic OH 149 – 145.2 2.25 ± 0.13 1 d o-disubstituted phenols 143.5 – 141.5 nd 0 catechols 140.5 – 138.3 5.30 ± 0.20 2.36 ± 0.05 noncatecholic o-substituted phenols 138.3 – 137.8 1.97 ± 0.05 0.88 ± 0.03 o-unsubstituted phenols 137.8 – 136.5 2.02 ± 0.08 0.90 ± 0.03 COOH 135.5 – 134.0 ndd 0 a Integration ranges adapted from Melone et al.39,40 b Ratio between the different OH groups, taking 1 for aliphatic OH as internal reference c Ratio between the different OH groups in catechin d nd = not detected


Theoretical ratioc 1 0 2 1 1 0

Page 33 of 46


Table 3. Comparison of the quantification of OH groups with different NMR techniques (averages and standard deviations are based on three distinct measurements)

Total aliphatic OH (mmol.g-1)

RSD (%)a

Total phenolic OH (mmol.g-1)

1

H NMR (acetylation) 3.46 ± 0.04 1.2 8.05 ± 0.09 H NMR (DMSO + D2O) ndb 10.45 ± 0.55 31 P NMR 2.25 ± 0.13 5.8 9.37 ± 0.34 a Relative Standard Deviation = Standard deviation / Average ratio b nd = not detectable with this measurement 1


RSD (%)a 1.1 5.3 3.7


Page 34 of 46

Table 4. Comparison of the main structural information determined for Acacia catechu tannins with various condensed tannins

Genus

Specie

Major flavanol unit

Acacia

Acacia catechu procyanidin Acacia auriculiformis prorobinetinidin Acacia mangium prorobinetinidin Acacia confuse procyanidin Acacia mearnsii prorobinetinidin Schinopsis lorentzii and Schinopsis profisetinidin Schinopsis balansae Pinus Pinus pinaster procyanidin Pinus radiata procyanidin Pinus maritimus procyanidin / prodelphinidin Pinus brutia procyanidin / prodelphinidin a Maximum DP Measured by MALDI-TOF MS


maximum DPa 4 10 11 11-12 8

Reference 34 35 36 31

10

31

13 15 20-21 6

61 61 62 45

Page 35 of 46


Figures

Figure 1



Figure 2


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Figure 3



Figure 4


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Figure 5



Figure 6


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Figure 7



Figure 8


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Figure 9



Figure 10


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Figure 11



Table of content image


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Characterization and Physicochemical Properties of Condensed

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