Soil Science and Plant Nutrition

ISSN: 0038-0768 (Print) 1747-0765 (Online) Journal homepage: http://www.tandfonline.com/loi/tssp20

Behavior of phenolic substances in the decaying

process of plants: I. Identification and Quantitative
Determination of Phenolic Acids in Rice Straw and
Its Decayed Product by Gas Chromatography
Shozo Kuwatsuka & Haruo Shindo
To cite this article: Shozo Kuwatsuka & Haruo Shindo (1973) Behavior of phenolic substances
in the decaying process of plants: I. Identification and Quantitative Determination of Phenolic
Acids in Rice Straw and Its Decayed Product by Gas Chromatography, Soil Science and Plant
Nutrition, 19:3, 219-227, DOI: 10.1080/00380768.1973.10432591
To link to this article: http://dx.doi.org/10.1080/00380768.1973.10432591

Published online: 29 Mar 2012.

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Soil Sci. Plant Nutr., 19 (3), 219-227, 1973

BEHAVIOR OF PHENOLIC SUBSTANCES IN THE DECAYING

PROCESS OF PLANTS

I. Identification and Quantitative Determination of Phenolic

Acids in Rice Straw and Its Decayed Product
by Gas Chromatography
Shozo KUWATSUKA and Haruo SHINDO
Faculty of Agriculture, Nagoya University, Nagoya, Japan
Received April 2, 1973
Phenolic acids in rice straw and its decayed product were surveyed and
quantitatively analyzed by gas chromatography.
1) Thirteen kinds of phenolic acids in rice straw and its decayed product were
identified. Besides P-hydroxybenzoic, vanillic, P-coumaric, and ferulic acids which
had been already reported, nine phenolic acids were newly identified; these were
benzoic, salicylic, syringic, protocatechuic, j9-resorcylic, caffeic, sinapic, gallic, and
gentisic acids.
2) A gas chromatographic analysis was applied to the micro-determination of
major phenolic acids in rice straw and .the decayed products. The methanolic
alkaline extracts from them were washed with ether, acidified, transferred into
ether, trimethylsilylated, and injected into a gas chromatograph equipped with a
silicon SE-30 column. The recoveries of P-coumaric, p.hydroxybenzoic, vanillic,
and syringic acids were more than 90%, and ferulic and syringic acids were re
covered at about 70%.
3) The content of each of the above described major phenolic acids in rice
straw ranged from 0.002 to 0.037% per dry weight. p.Coumaric acid was contained
in the largest amount. Ferulic and vanillic acids followed. In the decayed straw,
these contents decreased to 0.002-0.017%. The whole phenolic substances in ether
extracted fraction were present at 0.34%, and the amount decreased to one-third
during the decaying process.

Recently, rice straw has been applied back in larger amounts onto paddy and also
upland fields, especially for green house croppings, as an organic material mainly for
soil improvement. The degradation products of rice straw in the soils may influence
the growth of crops in both nutritional and physiological aspects. The elucidation of
the mechanism of humus formation from rice straw is also of importance for understanding its influence on plant growth.
Phenolic substances are widely distributed in various plants, including the rice
plant. Some of the substances, which enter into soils from the plants, cause dieback
219

220

S. KUWATSUKA and H. SHINDO

disease or other abnormal growth as inhibitors against plant growth. It was reported
that p-coumaric acid and other phenolics, for instance, inhibited the growth of the
upland rice plant (1 ), pea ( 2 ), and sugarcan.;: ( 3 ), and that isoflavones caused the
abnormal growth of red clover ( 4 ). Moreover, phenolic moieties are included in the
chemical structure of humic acids ( 5, 6 ).
NORD ( 7) found phenolic compounds such as ferulic acid, vanillic acid, dehydrodivanillin, and vanillin in the products from lignin which had been degraded by white
rot fungi. WHITEHEAD ( 8) detected phenolic compounds such as p-hydroxybenzoic,
vanillic, p-coumaric, and ferulic acids from soils. WANG, et al. ( 9) also found phenolic
acids in some Taiwan soils in which the yield of sugarcane decreased after multiple
planting. On the other hand, KUWATSUKA and OSHIMA ( 10) isolated and/or identified
p-hydroxybenzoic, vanillic, p-coumaric, and ferulic acids from rice leaves. INAMATSU
( 11 ) also found P-coumaric acid in a methanol extract of rice straw and recognized
that the amount of the acid decreased during the heaping of rice straw.
Most of these reports are deficient in quantitative data for these phenolic substances. Further quantitative information is necessary to elucidate the behavior of
phenolic substances in the decaying process of rice straw in soils and in compost.
In this paper, phenolic acids in rice straw and its decayed product were surveyed
in more detail and quantitatively analysed by gas chromatography.
MATERIALS AND METHODS
Rice straw of the "Kinmaze" variety was air-dried and crushed by a 'Wiley's
crusher so as to pass through a 2 rom sieve. For the preparation of the decayed
product, the crushed straw was put in beakers, brought to 60% water content, covered
with Saran Wrap, and incubated at 50oc for 45 days. The samples were mixed well
once a week.
Phenolic aldehydes and acids were obtained commercially, including p-hydroxybenzaldehyde, vanillin, resorcinol, orcinol, p-coumaric acid, ferulic acid, syringic acid,
protocatechuic acid, .S-resorcylic acid, caffeic acid, sinapic add, gallic acid, gentisic
acid, p-hydroxybenzoic acid, benzoic acid, vanillic acid, and salicylic acid.
Gas chromatography was carried out on a Hitachi Gas Chromatograph Model 063
equipped with a flame ionization detector, and dual stainless columns, 2 mx3 rom (inner
diameter), packed with chromosorb W (60-80 mesh) coated with 1.5% Silicone SE 30
(Shimazu). The flow rate of the carrier gas (nitrogen) was 30 ml/min. The temperatures of injector and detector were 280 and 300C, respectively. The column temperature was programmed from 100 to 250C at 5C/min. For standard calibration curves,
the ratio of the peak height of phenolic acid to that of the internal standard substance,
p-chlorobenzoic acid, was plotted to the ratio of the amount of the former to a definite
amount of the latter.
Identification of phenolic acids in rice straw and its decayed product.
Fifteen grams
each of the crushed rice straw sample or the decayec\. product was suspended in 1 liter
of methanolic sodium hydroxide (methanol : 0.1 N Na0H=7 : 3) and shaken for 30 min.
The extract solution was centrifuged and then filtered. The filtrate was adjusted to
pH 7.Q-7.5 with dil. HCI, concentrated to about 300 ml at 45-50C in a water bath.
and acidified to pH<2 with dil. HCl. The solution was extracted with 300m! of ether

Behavior of Phenolics in Decaying Process of Plants (I)

221

three times. The ether was dried over anhydrous Na 2SO., and evaporated to dryness
below 45C. To the residual sample was added 0.3 ml of TMS (N, 0-bis-trimethylsilyl
acetamide, 25% solution in acetonitrile), and the solution was allowed to stand for 3
min in heat below 60C and was then injected into the gas chromatograph. The individual phenolic compounds in rice straw and the decayed product were identified with
the authentic chemicals by co-chromatography,
Recovery test 1 (Recovery from methanolic NaOll solution).
Phenolic acids of the
various concentrations shown in Table 2 were added to 100 ml of the methanolic sodium
hydroxide above described. The solution was adjusted to pH 7.0-7.5 with dil. HCl,
evaporated to 30 ml at 45-50C in a water bath and acidified to pH <2 with dil. HCI.
'The phenolic acids were extracted with 100 ml of ether four times. The ether solutions
were combined, dried with anhydrous sodium sulfate, and then concentrated to a small
volume. The materials were completely transferred to a small vial with washing
acetone. P-Chlorobenzoic acid as the internal standard substance was added into the
vial and the solvent was evaporated off below 60C. The sample with added 100 pl of
'TMS solution was allowed to stand for 3 min in heat below 60C and injected into
the gas chromatograph. From the ratio of the peak heights of the samples to that of
the internal standard substance, the amount of each phenolic acid was obtained by the
standard calibration curves which had been previously prepared for the determination
of the amount of phenolic acids.
Recovery test 2 (Recovery from the rice straw with added authentic chemicals).
The
-crushed straw was extracted with acetone and methanolic sodium hydroxide successively, and then the residue was washed and air-dried. To 5 g of the residue, 15 ml for a
wet condition or 50 ml for a flooded condition of distilled water and 5 to 10 ml of a
methanol solution of phenol carboxylic acids of the various amounts shown in Table 3
were added and allowed to stand for 30 min at room temperature. After the addition
of 150 ml of the methanolic sodium hydroxide solution, each sample was shaken for 30
min. The suspension was centrifuged at 3,000 rpm for 30 min and filtered. This
procedure was repeated three times. The filtrates were combined and made up to 500
ml. The subsequent procedure was as described above (Recovery test 1). Moreover,
as the blank test, phenolic acids in the straw sample with no authentic chemicals added
were also determined by the same procedure. The recovery of phenolic acids was cal-culated by the following formula:
Recovery (%) =

B-C
--x-

100

where A is the added amount of phenolic acids, B the recovered amount from the
straw sample with the added acids, and C the recovered amount from the sample
without added acids.
Determination of major phenolic acid components in rice straw and the decayed straw.
Each content of major phenolic acids in 5 g of the straw sample was determined by
applying the procedure of "Recovery test 2" before and after the incubation at sooc for
45 days under the wet condition.
Determination of the total phenolic substances in the ether-extractable acid fraction
.()f rice straw and in that of the decayed product.
Total phenolic substances in the

S. KUWATSUKA and H. SHIN DO

222

ether-soluble acid fractions derived from the concentrated methanol-NaOH extracts, described in "Recovery test 2" were also determined by a modification of the method of
Folin et al. ( 12 ). After the ether solution was dried up, 50 ml of 20% Na2C08 and
10 ml of Folin's reagent were added to the residue. The mixture was filled up to 100
ml with distiUed water and allowed to stand for 20 min at room temperature and the
optical density was measured at 700 mp. The amount of phenolic substance was calculated from the optical density of p-coumaric acid, which was present in the largest
amount of the phenolic acids in rice straw.
RESULTS AND DISCUSSION

Phenolic acids in rice straw and the decayed product were surveyed by the gas
chromatographic method as described above. As a result, nine phenolic acids were
newly detected by co-chromatography with authentic chemicals in addition to P..hydroxybenzoic, vanillic, p..coumaric, and ferulic acids which had been already identified as
phenolics in rice plants ( 10 ).
The gas chromatogram of phenolic acids in rice straw is shown in Fig. 1. The
100
14

..
"'

..,2.

11

so

!:E:

10

20
Retention time

6n in)

30

40

Fig. 1. A gas chromatogram of phenolic acids in rice straw.

The numbers in the chromatogram show the numbers of
the phenolic acids in Table 1.

major phenolics were p-hydroxybenzoic, vanillic, p..coumaric, ferulic, salicylic, and

syringic acids. Benzoic, gallic, caffeic, sinapic, gentisic, protocatechuic, and ~-resorcylic
acids appeared as minor components. Protocatechuic and ~-resorcylic acids were identified by .TLC on a silica gel plate developed with chloroform : acetic acid (9 : 1) and
benzene : ethylacetate (6 : 4), because their peaks appeared at the same retention time
in gas chromatography (Fig. 1). Chemical structures and the relative retention times
of identified substances are shown in Table 1. p..Hydroxybenzaldehyde, vanillin, resorcinol, and orcinol were not detected.
In order to determine the accurate amount of phenolic acids, the internal standard

223

Behavior of Phenolics in Decaying Process of Plants (I)

Table 1. Chemical structures and the relative retention times of identified phenolic acids.
No

Phenolic acid

Benzoic
Salicylic
P.Hydroxybenzoic
Vanillic

2
3

13

Gentisic
Protocatechuic
JlResorcylic
Syringic
p-coumaric
Gallic
Ferulic
Caffeic
Sinapic

14

p-el-benzoic

()

1
8
9

10
11

12

Chemical structure
Benzoic acid
2-Hydroxybenzoic acid
4-Hydroxybenzoic acid
3-Methoxy-4-hydroxybenzoic acid
2, 5-Dihydroxybenzoic acid
3, 4Dihydroxybenzoic acid
2, 4-Dihydroxybenzoic acid
3, 5-Dimethoxy-4-hydroxybenzoic acid
4Hydroxycinnamic acid
3, 4, STrihydroxybenzoic acid
3-Methoxy-4-hydroxycinnamic acid
3, 4-Dihydroxycinnamic add
3, 5Dimethoxy-4-hydroxycinnamic acid
Internal standard substance
4..Chlorobenzoic acid

Relative reten
tion time
0.67

1.26
1.59

1. 91
2.00

2.05
2.05
2.15
2.30
2.34

2.61
2.66
2.90
1.00

method was used for the gas chromatography, p..Chlorobenzoic acid was used as the
internal standard substance because the peak did not interfere with compounds derived
from rice straw and its decayed product.
Standard calibration curves of major phenolic acids are shown in Fig. 2, in which
the linear relation is shown between the ratio of the peak height and that of the
.amount. Recovery of individual phenolic acid in "Recovery test 1" is shown in Table
2 and the average of recoveries were as follows: P..hydroxybenzoic acid 97%, vanillic
.acid 99%, p-coumaric acid 87%, ferulic acid 68%, salicylic acid 99.5%, and syringic
.acid 81%, respectively. Each recovery of these acids showed almost the same value
for the various concentrations of phenolic acids added. The recovery of ferulic acid
was lower than those of the other phenolic acids, It seemed to be caused by the
structural change of the acid during the procedure and/or by the long retention time,
Recovery of individual phenolic acids in "Recovery test 2.. is shown in Table 3.
Both recoveries by "test 1" and "test 2" were almost same. This means that the
:Phenolic acids added were mostly recovered from the straw sample by extraction three
times with methanolic sodium hydroxide. The outline of quantitative determination is
shown in Fig. 3. The determined values of phenolic acids in rice straw and the decayed
product were as follows: p-coumaric acid 0.037%, 0.017%, ferulic acid 0.022%, 0.013%,
vanillic acid 0.012%, 0.009%, and P..hydroxybenzoic acid 0.002%, 0.002%, per dry weight,
respectively (Table 4). The content of p-coumaric acid was the largest of all phenolic
acids. The amount of other phenolic acids, except for p.hydroxybenzoic acid, decreased
during the decaying process of the straw, These amounts of individual acids and the

S. KUWATSUKA and H. SHINDO

224

.g

""."'
Ill

""!!c:

...f
Ill

.::::

1.6
1.4
1.2

1.0

} 0.8

"'

.:&, 0.6

......o;
:

... 0.4
0

0.2

0o

0.2
0.4
0.6
0.8
1.0
1,.2
1.<&
Ratio of amount (sample/internal standard sub.)

Fig. 2. Standard calibration curves of major phenolic acids.

0, p-hydroxybenzoic acid ; e, vanillic acid ; X, p-coumaric
acid; 0, ferulic acid.
Table 2. Recovery of individual phenolic acid in Recovery test 1.
Phenolic acid
p-Hydroxybenzoic

Vanillic

p-Coumaric

Ferulic

Salicylic
Syringic

Added amount
(pg)

Recovered amount
(pg)

Recovery
(%)

382
153

377
144

99

76

75

406

406

98
100

162

155

96

81
338

81

100

304

90

135

118

68

57

87
84

364

258

146

98

71
67

73

48

66

402

402

100

161

159

99

412

346

84

165

127

77

94

Average;of recovery
(%}

97

99

87

68

99,5
81

Behavior of Phenolics in Decaying Process of Plants (I)

225

Table 3. Recovery of individual phenolic acid in Recovery test 2.

Phenolic acid

Condition 1>

Added
amount
(pg)

Blank
(no addition)
(pg)

Recovered
amount

Recovery
(%)

Average of
recovery
(%)

p-Hydroxybenzoic

804

Trace

737

92

91

804

724

90

784

876

93

520

95

392

508

92

796

2696

93

2303

88

398

2326

94

776

1431
1158

70
70

1144

66

555

74

529

71

792

97

790

97

388

95

Salicylic

w
w
F

p-Coumaric

w
w
F

w
w

Ferulic

F
Syringic
Vanillic

1)

392

147

1952

398

888

388
388

Trace

748

748
816

816

408

+2>

W, Wet condition. F, Flooded condition.

2)

(pg)

93

92

69
73

96

+, <50pg.

Table 4. Content of phenolic substances in rice straw and the decayed product.
Decayed product

Rice straw
Phenolic substance

Content
(pg)

%per
dry wt.

Content
(pg)

Ratio to ini% per dry

tial amount wt. of decayed sample
(1o)

1642

0.037

446

27.2

0.017

Ferulic acid

981

0.022

340

34.6

0.013

Vanillic acid

543

0.012

240

44.2

0.009

79

0.002

63

79.7

0.002

Salicylic acid

<SO

<0.002

<SO

Syringic acid

<SO

<0.002

<SO
5.7 mg

p-Coumaric acid

p-Hydroxybenzoic acid

Total ether-extractable phenolics

1S.1 mg

0.34

<0.002
<0.002
37.7

0.28

amounts of total phenolic substances in the ether-soluble acid fractions by Folin's

colorimetric determination were compared (Table 4). Although the content of p-coumaric
acid was almost half of the total amounts of the 4 major phenolic acids contained both
in non-decayed and decayed straws, the total amounts of these 4 phenolic acids were

S. KUWATSUKA and H. SHINDO

226

Rice straw or decayed straw

alkaline methanol
shake for 30 min

Centrifuge (3, 000 rpm)

Filter

Fill up to 500 ml

pH 7.0-7.5 (dil. HCl)

Concentrate

washing with ether

Residual fraction

Same as left column

pH<2 (dil. HCl)

Ether extract (ether-soluble ac1d

fraction)
anhydrous sodium sulfate

Concentrate

Dry up

transfer to a small vial

Na2COa

internal standard substance

Folin reagent
distilled water

Evaporate off

Stand for 20 min

TMS

Colorimetry (700 mp)

Gas chromatography
Fig. 3. The outline of the quantitative determination.

only 21 and 19% of the whole amounts of ether-soluble phenolic substances which were
calculated as p-coumaric acid, in non-decayed straw and the decayed product, respectively. This result suggests that either rice straw or the decayed product contains
many other phenolics besides these 4 major phenolic acids, as is shown by the gas
chromatogram in Fig. 1. These percentages also suggest that the ether-soluble acid
fraction contains other types of phenolic compounds such as tricin, an ether-soluble
flavonoid compound, which had been isolated from rice leaves (13), and these compounds were not detected by this gas chromatographic method. Further, the ratio of
the summed amounts of the major phenolic acids to the amount of the total phenolic

Behavior of Phenolics in Decaying Process of Plants (I)

227

substances in the ether-extracted fraction was only slightly changed, although both
amounts decreased to about one-third of the initial amounts after the incubation of
straw for 45 days. This result may suggest that considerable amounts of various
ether-extractable phenolics in rice straw behaved in a similar manner during the in
cubation, although these phenolics were thought to be decomposed or polymerized to
other molecules and produced from substances of higher molecular weight during this
decaying process. And, it may be said that the decomposition or polymerization of
these phenolics of low molecular weight was more rapid than their production from
other ether-unextractable substances such as lignin.
Acknowledgement. Special thanks are due to Prof. K. Kumada of the authors' laboratory for
his guidance and assistance during the course of this study.
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