ANTIOXIDANT POTENTIAL OF HYDROLYZED POLYPHENOLIC ANTIOXIDANT POTENTIAL OF HYDROLYZED POLYPHENOLIC EXTRACTS FROM TARA (Caesalpinia spinosa) PODS EXTRACTS FROM TARA (Caesalpinia spinosa) PODS

Flor Chambi a,b, Rosana Chirinos a, Romina Pedreschi c, Indira Betalleluz-Pallardel a, Frédéric Debaste b, David Campos a(*)

a Instituto de Biotecnología (IBT), Universidad Nacional Agraria La Molina-UNALM. Av. La Molina s/n, Lima, Perú b Transfers, Interfaces and Processes (TIPs), Université Libre de Bruxelles-ULB. 50 avenue F.D. Roosevelt, C.P. 165/67, 1050 Brussels, Belgium c Fresh, Food & Chains. Wageningen UR Food & Biobased Research. Bornse Weilanden 9, 6708WG, The Netherlands

(*) E-mail address: dcampos@lamolina.edu.pe

INTRODUCTION

Tara (Caesalpinia spinosa), a leguminous tree from Peru has traditionally and extensively been used by

Peruvian folk medicine. Especifically tara pods have been consumed and used as infusions. Tara pods

contain gallotannins (~ 50% w/w), which are hydrolysable tannins and are considered to be one of the most

potent antioxidants known from plant sources. Gallotannins hydrolysis yields either gallic acid or tannin

oligomers, which display even higher antioxidant capacity than tannins.

This study aims: (1) to study the chemical hydrolysis of gallotannin extracts from tara, (2) to evaluate the

hydrolsis degree (HD) of tara extracts regarding in vitro antioxidant capacity and (3) to evaluate the

performance of these extracts by differential scanning calorimetry to retard soybean oil oxidation.

MATERIALS AND METHODS

Fresh tara pods were purchased from a local market in Caraz

(Ancash, Peru). Tara pods (without seeds) were washed and

air-dried at 55 ºC until a final humidity of ~ 3 % was reached.

Tara pods minced

Extraction

Hydrolysis

whole extract (WE)

80 % (v/v) acetone/water

QUANTITATIVE ANALYSIS

RESULTS

Fig. 1. Total phenolics ( ) and gallic acid ( ) (a), and hydrolysis degree

(b) evolution during chemical hydrolysis of tara extracts (with 2N

H2SO4, 20 mg of GAE/mL and 100ºC). Different capital and short letters on each

curve indicate significant differences (p < 0.05) as revealed by a Duncan test

Fig. 2. Specific antioxidant capacity by ABTS (a), FRAP (b) and ORAC (c)

assay and liphophility (d) evolution during chemical hydrolysis of tara

extracts (with 2N H2SO4, 20 mg of GAE/mL and 100ºC). Different capital and

short letters on each curve indicate significant differences (p < 0.05) as revealed by a Duncan

test.

CONCLUSIONSThis study provides strong evidence about the antioxidant potential of TPHEs with different hydrolysis degree. An increase in vitro antioxidant capacity for the different TPHEs was observed as the hydrolysis degree increased, especially from 4 h de hydrolysis. At a concentration of 100 ppm TPHEs at 9 and 20 h hydrolysis significantly better antioxidant protection against soybean oil oxidation was observed than with 100 ppm TBHQ using the scanning calorimetry assay. These results indicate that 4 and 9 h of chemical hydrolysis of tara extract under the tested conditions are sufficient to obtain a product with good antioxidant properties to be utilized as an alternative source of natural antioxidants.

This research was supported by the Peru Biocomercio project (Peru) and by the PIC project of the Belgian Coopération Universitaire au Développement (CUD, Belgium).

REFERENCES- Arnao, M., Cano, A., & Acosta, M. (2001). Food Chemistry, 73, 239-244. - Benzie, I., & Strain, J. 1996. Analytical Biochemistry, 239, 70-76.- Chirinos, R., Campos, D., Costa, N., Arbizu, C., Pedreschi, R., & Larondelle, Y. (2008). Food Chemistry, 106, 1285–1298.- Inoue, K.H., & Hagerman, A.E. (1988). Analytical Biochemistry, 169, 363-369.- Ou, B., Hampsch-Woodill, M., & Prior, R. (2001). J. Agric. Food Chem., 49, 4619-4626.- Salminen, J. P. (2003). Journal of Chemical Ecology, 29, 1289-1305. -Singleton, V., & Rossi, J. (1965). Am. J. Enol. Vitic., 16, 144-158.- Takacs-Novak K, Avdeef A (1996).. J Pharm Biomed Anal 14: 1405–1413.- Tan, C., Chen, Y., & Selamat, J. (2002). Food Chemistry, 76, 385-389.

ACKNOWLEDGMENTS

H2SO4

final 2N H2SO4 concentration;100ºC

0-28 hours

Liquid-liquid partitionethyl acetate

aqueous phase

hydrolyzed extracts (HE)

Vacuum concentration

organic phase

ethanol

tara purified and hydrolyzed extract (TPHE)

EXTRACTS PREPARATION

- Total phenolics compounds. Method of Folin Ciocalteau

(Singleton and Rossi ,1965)-Gallic acid and gallotannin contents. Rhodanine assay

(Inoue and Hagerman,1988; Salminen, 2003). - In vitro Antioxidant Capacity. ABTS, FRAP and ORAC

assays (Arnao et al., 2001; Benzie and Strain,1996; Ou et

al., 2001). - Differential Scanning Calorimetry (DSC) Assay. Isothermal

temperature at 140 ºC (Tan et al., 2002).- Lipophicity was examined by measuring the partition

coefficients using an n-octanol/aqueous system (Takàcs-

Novàk and Avdeef, 1996). -HPLC-PAD Analysis of Phenolic Compounds. Phenolic

profiles were determined (Chirinos et al.,2008).

0

5

10

15

20

25

0 5 10 15 20 25 30

Time (h)

Total phenolic compunds

Free gallic acid

a

G

ABC

DD

E

F

HI J I I

JK K

ab ab ababb abb b b b ab ab

Gal

lic a

cid

or p

heno

lic c

omp

ound

s co

ncen

trat

ion

(mg

GA

E/m

l) aa

(a)

0

20

40

60

80

100

120

0 5 10 15 20 25 30

Time (h)

a

(b)

bHyd

rols

isde

gree

(%)

(b)

cdde

f

g

h i j jkh h

k

100

200

300

400

500

600

Ant

ioxi

dant

cap

acity

(μm

ol T

E/m

L)

a

b

c

d

de e

f g

(a)

0

5

10

15

20

25

30

0 5 10 15 20

Spec

ific

ant

ioxi

dant

cap

acity

(μm

ol T

E/m

g G

AE

)

Time (h)

a

bc

d d d d d d(b)(a)

0

100

200

300

400

500

600

Ant

ioxi

dant

act

ivity

(u

mol

trol

ox e

qui./

ml)

Time (h)

ab

ccd cdede efef f

(a)

0

5

10

15

20

25

30

0 5 10 15 20

Spec

ific

ant

ioxi

dant

act

ivity

( μm

ol T

E /

mg

GA

E)

Time (h)

aa

bc c c c c c

(b)

0.00

0.10

0.20

0.30

0.40

0.50

AU

0.00

0.10

0.20

0.30

0.40

0.50

Retention times (min)0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 90.00 100.00

d

c

b

a

AU

0.00

0.10

0.20

0.30

0.40

0.50

AU

0.00

0.10

0.20

0.30

0.40

0.50

AU

0.00

0.10

0.20

0.30

0.40

0.50

d

c

b

aG

allic

acid

Gal

licac

idG

allic

acid

0.00

0.10

0.20

0.30

0.40

0.50

AU

0.00

0.10

0.20

0.30

0.40

0.50

Retention times (min)0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 90.00 100.00

d

c

b

a

AU

0.00

0.10

0.20

0.30

0.40

0.50

AU

0.00

0.10

0.20

0.30

0.40

0.50

AU

0.00

0.10

0.20

0.30

0.40

0.50

d

c

b

aG

allic

acid

Gal

licac

idG

allic

acid

Fig. 3. Chromatograms of hydrolyzed tara extracts at different hydrolysis degrees: 0 h (non hydrolyzed extract) (a), 4 h (38.8% HD) (b), 9 h (93.7% HD) and 20 h (100% HD).

0

50

100

150

200

Ant

ioxi

dant

act

ivity

(u

mol

trol

ox e

qui./

ml)

Time (h)

aa a b

aba ba ab

(a)

0

2

4

6

8

10

0 5 10 15 20

Spec

ific

ant

ioxi

dant

act

ivity

( μm

ol T

E /

mg

GA

E)

Time (h)

aba ab b a a a ab ab

(b)(c)

0,0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

0,8

0,9

1,0

0 5 10 15 20

log

P o

w

Time (h)

a

b

c cdd d d

dd

(d)Table 1. Induction periods (IP) and stabilization factors (SF) for TPHEs at different hydrolysis times using differential scanning calorimetry

a Results are means SD (n=3). Means within a column with the same letter are not significantly different (p >0.05) as revealed by a Duncan test.

HydrolysisTime(h)

Induction period(min)

Stabilization factor

0.0 40.17 ± 0.28 b 1.07 ± 0.01 b

0.5 42.03 ± 0.37 c 1.12 ± 0.01 c

1.5 43.35 ± 0.36 d 1.16 ± 0.01 d

4.0 47.27 ± 0.29 e 1.26 ± 0.01 e

5.0 47.67 ± 0.29 e 1.27 ± 0.00 e

6.0 48.38 ± 0.13 f 1.29 ± 0.00 f

8.0 49.11 ± 0.04 g 1.31 ± 0.00 g

9.0 50.42 ± 0.18 h 1.35 ± 0.01h

20.0 50.24 ± 0.41 h 1.34 ± 0.01 h

TBHQ 49.45 ± 0.25 g 1.32 ± 0.00 g

Control 37.35 ± 0.19 a 1.00 ± 0.00 a