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by elicitors-induced on greenhouse condition.

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1 by elicitors-induced on greenhouse condition.
Antioxidant activity and major isoflavonoid contents of White Kwao Krua  by elicitors-induced on greenhouse condition.

2 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.

3 White Kwao Krua Found in northern and north eastern.
[Pueraria candollei Grah. var. mirifica (Airy Shaw et. Suvatabandhu) Niyomdham]. Found in northern and north eastern. It is considered to be a rejuvenating drug and has a history in folk medicine. Name “White Kwao Krua” was definitively identified as Pueraria candollei in 1952 from which an unusual estrogenic. White Kwao Krua (WKK) has a high concentration of phytoestrogen.

4 Applications cosmetic industry Pharmaceuticals industry Livestock
breast cream eye gel skin moisturizer. Pharmaceuticals industry Replacement hormones Loss bone metabolism Livestock Animals feed

5 Antioxidant activity Antioxidant compounds play an important role as a health-protecting factor. Antioxidants reduce the risk for chronic diseases including cancer and heart disease. The main characteristic of an antioxidant is its ability to trap free radicals. superoxide anion radical (O2) hydroxyl radical (OH) peroxyl radical (ROO) Cell metabolism & pollutions Gutteridge (1994)

6 Major Isoflavonoids Genistein Puerarin Igham et al. (1986)
Phytonutrient Manopause (hormonal replacement) Loss bone (bone metabolism) Puerarin Hypertension antioxidant Igham et al. (1986)

7 Elicitors Molecules that stimulate secondary metabolism
Elicitors may form inside or outside plant cells, and are distinguished as endogenously or exogenously inducers. Depending on their origin, they are classified as biotic or abiotic Heike and Dietrich (1995)

8 Elicitors Chitosan Soybean; ppm increased seed isoflavone % and Daidzein 150 % Tawaha et al. (2005) Salicylic acid Lupinus ruteus; µM increased Genistein 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)

9 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) Water salicylic acid (SA) CuCl2 CuCl2 + SA Chitosan Chitosan + SA Chitosan + CuCl2 Chitosan + CuCl2 +SA

10 Antioxidant determination by DPPH
Inhibitory concentration at 50% (IC50) Plot between % radical scavenging VS concentration of each treatment Equation of nonlinear regression

11 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 1 ml / 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) Standard isoflavonoids: puerarin (95 %) and genistein (99.9 %)

12 Result Effect on root growth Combinations % Moisture Diameter
Fresh weight Dry weight water 73.23 64.56 589.32 147.33 SA 74.65 62.07 611.93 152.98 CuCl2 72.53 77.53 591.30 147.83 CuCl2 + SA 77.20 72.30 561.38 140.35 Chitosan 75.97 68.83 614.32 153.58 Chitosan + SA 74.73 66.57 622.53 155.63 Chitosan + CuCl2 73.36 75.36 542.44 135.61 Chitosan + CuCl2 + SA 74.85 602.36 150.59

13 Antioxidant activity by DPPH

14 Major isoflavonoids by HPLC



17 Conclusion & Discussion
The results showed that all combinations had no statistically significant effect on the diameter, fresh weight, dry weight and moisture content of the tuberous roots. it had a statistically significant effect on antioxidant activity and the amount of genistein and puerarin. The combination of chitosan (1000ppm) + copper(II)chloride (300ppm) + salicylic acid (100ppm) gave the highest antioxidant activity (IC50 = 1.99 mg/ml by DPPH).

18 The elicitors did not have the bad effects on root growth of WKK.
Conclusion & Discussion The combination of chitosan (1000ppm) + copper(II)chloride (300ppm) gave the highest amount of genistein and puerarin were determined by HPLC are 31.9 g/100g in dry weight and g/g in dry weight, respectively. The elicitors can increase antioxidant activity and major isoflavonoids content in tuberous roots of WKK. The elicitors did not have the bad effects on root growth of WKK.

19 Thank you for your attention.

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