Antioxidant activity and major isoflavonoid contents of White Kwao Krua 

by elicitors-induced on greenhouse condition.

The experimental purpose

Study on the effect of using the elicitors on induce the antioxidant activity and the major isoflavonoids content in White Kwoa Krua in the greenhouse condition.

• F ound in northern and north eastern.• It is considered to be a rejuvenating d

rug and has a history in folk medicine.• N ame “White Kwao Krua” was definiti

vely identified as Pueraria candollei in 1952 from which an unusual estrogeni

c. White Kwao Krua (WKK) has a

high concentration of

phytoestrogen .

White Kwao Krua [Pueraria candollei Grah. var. mirifica (Airy Shaw et. Suvatabandhu) Niyomdham].

Applications

• cosmetic industry • breast cream• eye gel• skin moisturizer.

• Pharmaceuticals industry • Replacement hormones• Loss bone metabolism• Livestock

• Animals feed

Antioxidant activity • Antioxidant compounds play an imp

- ortant role as a health protecting factor.• A ntioxidants reduce the risk for chronic dise

ases including cancer and heart disease.• The main characteristic of an antioxidant is i

ts ability to trap free radicals.

Cell metabolism & pollutions superoxide ani

on radical (O2

) hydroxyl radic

al (OH) peroxyl radical

(ROO)

Gutteridge (1994)

Major Isoflavonoids

• Genistein– Phytonutrient– Manopause (hormonal replacement)– Loss bone (bone metabolism)

• Puerarin– Hypertension– antioxidant

Igham et al. (1986)

Elicitors Molecules that stimulate secondary

metabolism Elicitors may form inside or outside p

lant cells, and are distinguished as e ndogenously or e

xogenously inducers. D epending on their origin, they are c

lassified as biotic or abiotic Heike and Dietrich (1995)

Elicitors

Chitosan

Soybean; 1000

ppm increased seed isoflavone - 1696

%and Daidzein 15 0%

Tawaha et al. (2005)

Salicylic acid

Lupinus ruteus; 80 0 µM increased

Genistein 1212 µ g/g DW.

Kneer et al.

( 1999)

Copper (II)

chloride

- Pueraria montana; 100 ppm increased Genistein 5 folds- Pueraria lobata; 10 mM for 2 hour increased Genistein 10 folds

Takashi et al. (1990)Hakanatsuka (1991)

Material & Methods

• Plant Planting WKK in greenhouse

at SUT farm Selected 48 plant WKK were

age of 1 year old • Experimental plan

CRD 6 replication with 8 treatments

• Elicitor combination

(treatments)

Watersalicylic acid (SA)

CuCl2 CuCl2 + SA

ChitosanChitosan +

SAChitosan +

CuCl2 Chitosan + CuCl2 +SA

Antioxidant determination by DPPH

Inhibitory concentration at 50% (IC50) Plot between % radical scavenging VS concentration of each treatmentEquation of nonlinear regression

• Major isoflavonoids determination by HPLC

HPLC condition• HPLC column RP-C18 Agilent® (4.6 mm x

15 cm)• Detector Diode array 254 nm • Flow rate 1ml / min• Volume injection 10 µl• Presser pump 111 bar • Mobile phase (A): 0.1% (v/v) acetic acid in

water (B): 0.1% (v/v) acetic acid in

acetonitrile

Time (min) mobile phase (%)(A) (B)

0 90 1035 72 28

Standard isoflavonoids: puerarin (95 %) and genistein (99.9 %)

Result

CombinationCombinationss % Moist

ureDiameter

Freshweigh

t Dry w

eight

waterwater73.23

64.56

589.32

147.33

SASA74.65

62.07

611.93

152.98

CuCl2CuCl272.53

77.53

591.30

147.83

CuCl2 + SA CuCl2 + SA77.20

72.30

561.38

140.35

ChitosanChitosan75.97

68.83

614.32

153.58

Chitosan + S Chitosan + SAA 74.73

66.57

622.53

155.63

Chitosan + C Chitosan + CuCl2uCl2 73.36

75.36

542.44

135.61

Chitosan + C Chitosan + C uCl2 + SA uCl2 + SA 72.53

74.85

602.36

150.59

Effect on root growth

Antioxidant activity by DPPH

3.44

2.362.60

2.312.62

2.43 2.49

1.99

0.070.00

0.50

1.00

1.50

2.00

2.50

3.00

3.50

4.00

Elicitors

IC50

(m

g/m

l)Antioxidant activity by DPPH

Major isoflavonoids by HPLC

min26.5 27 27.5 28 28.5 29 29.5

mAU

0.5

1

1.5

2

2.5

3

3.5

DAD1 A, Sig=254,4 Ref=360,100 (RUAM\GCTRT008.D) 2

6.43

2 27.

008

28.

227

29.

657

Isoflavonoids content

31.27

66.54 67.39

46.98 44.93

72.82

106.30

74.78

2.1

12.9

9.8 11

.3 13.4 16

.3

31.9

8.1

0.00

20.00

40.00

60.00

80.00

100.00

120.00

water SACuCl2

CuCl2 + SA

Chitosan

Chitosan + SA

Chitosan + CuCl2

Chitosan + CuCl2 + SA

Elicitors

unit

puerarin (ug/g DW)

genistein (ug/100 g DW)

• The results showed that all combinations h ad no statistically significant effect on the

diameter, fresh weight, dry weight and mo isture content of the tuberous roots.

• it had a statistically significant effect on a ntioxidant activity and the amount of geni

stein and puerarin.

• The combination of chitosan (1000ppm) + copper(II)chloride (300ppm) + salicylic aci

d (100ppm) gave the highest antioxidant a ctivity (IC50 = 1.99 mg/ml by DPPH).

Conclusion & Discussion

• T 1000he combination of chitosan ( ppm) gggg ggg gg+ () (3 0 0 )

ggggg gg gggg gg ggggggggg ggg gggggggg g ggg 319determined by HPLC are . /1 0 0

1063in dry weight and . g/gindrywei ght , r esp .

• Tgg ggggggggg ggg gggggggg ggggggggggg gggggggg ggg ggggg ggggggggggggg ggggggg gg ggggg ousr oot s of WKK.

• The elicitors ggg ggg gggg ggg ggg ggggggg gg g gggggg gg gggg.

Conclusion & Discussion

Thank you for your attention.