estimates of total phenolics content in peruvian …

ESTIMATES OF TOTAL PHENOLICS CONTENT IN PERUVIAN ECOTYPES OF

LUPIN (Lupinus mutabilis) (BITTER AND DEBITTERED)

Zuly Sabelino-Francia1A, Patricia Glorio-Paulet1

B, Luis Rodriguez-Saona2C, Felix Camarena 1D

1 Universidad Nacional Agraria La Molina, Perú. 2 Ohio State University. USA.

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Objective

• Characterize the total phenolic content in 52 different Ecotypes of Peruvian Lupinus mutabilis.

• Evaluate the effect of water washing processing (for alkaloid removal) in the phenolics content that remains in ecotypes.

• Evaluate alternative methodologies in the phenolics content determinations, that could help in the characterization of thousand of ecotypes for Peruvian biodiversity. For example methods based in spectroscopy.

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Benefit of Phenolic Compounds.

Beneficial effects of phenolic compounds:

Antioxidant activity

Estrogen antagonism

Antiangiogenic effect.

Promotion of apoptosis

Inhibition of Cyt. P450, protein kinases and telomerases.

• They are powerful inhibitors of HNO2 (nitrous acid) which decomposes nitrosamines and causes deamination of DNA bases, especially guanine.

• Phenolic compounds protect the gastrointestinal system in situations of excessive production of reactive nitrogenous species

Fuente: Cadenas y Packer, 2002. Handbook of Antioxidants. Second Edition. CRC Taylor y Francis.

Phenolic Compounds in plants

Materials and MethodsFor this research, 52 ecotypes of Lupinus mutabilis (called “Chocho” or “Tarwi” in Peru) were obtained as seeds.

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6

52 Ecotypes studied

Fuente: Fotos tesis Ingeniero UNALM 2019 Autor ZulySabelino-Francia

Provided by: Legume Program at UNALM Peru

These seeds were stored in polietilene

bags under refrigeration at 5 °C until

analysis.

Weigh

Soaking

Cooking

Washing

Drying

Defatted

Grinding

Selection

Bitter seeds

Weigh

Defatted

Grinding

Bitter Tarwi

Flour

Debittered Tarwi

Flour

Seeds:hexane

1:35

Seeds:hexane

1:35

Seeds:water

1:3

Seeds:water

1:3

Seeds:water

1:6 12 h

5 days

2 periods

of 30 min

50°C 18h

Total Phenolics colorimetricdetermination

Phenolics extraction in 50% ethanol solution

as a solvent

Defatted tarwi flour (g): solvent (ml) 1:20. Sonicated twice in an

ultrasound bath, period = 30 min.

The quantification of the phenolic

content was performed by the Folin Ciocalteau Microplate format: readings at

726 nm

samples were centrifuged at 3000 RPM for 15 minutes at 5 ° C.

Quiroz-Reyes, C., M. Aguilar-Méndez, et al. (2013). "Comparative study of ultrasound and maceration techniques for the extraction of polyphenols fromcocoa beans (Theobroma cacao L.)." Revista mexicana de ingeniería química 12(1).

0

100

200

300

400

500

600

T1

T2

T3

T4

T5

T6

T7

T8

T9

T1

0T

11

T1

2T

13

T1

4T

15

T1

6T

17

T1

8T

19

T2

0T

21

T2

2T

23

T2

4T

25

T2

6T

27

T2

8T

29

T3

0T

31

T3

2T

33

T3

4T

35

T3

6T

37

T3

8T

39

T4

0T

41

T4

2T

43

T4

4T

45

T4

6T

47

T4

8T

49

T5

0T

51

T5

2

Phen

olic

toat

al C

onte

nt

(mg g

allic

acid

/100g)

Ecotype codeBitter Lupinus mutabilis Debittered Lupinus mutabilis

Figure 1. Phenolic Total Content in bitter and debittered Lupinus mutabilis

Fuente: tesis Ingeniero UNALM 2019 Autor Zuly Sabelino-Francia

Bitter* Debittered*

Mean 489.71 147.31

Minimum 428.49 94.45

Maximum 526.80 204.67

Standard deviation 21.66 28.61

Table 1. Average phenolic total content in genotypes of Lupinus mutabilis

* mg gallic acid / 100g sample

Ecotype OriginMin PLGO 3 JunínMax Compuesto blanco semi precoz Junín

70% reducción(Effect of water

washingdebittering)

35.8%

61.2%

For low Phenolics content genotypes, values ofreductions simlar to earlier reports of Jimenez-Martinezet al 2007


Construction of MIR spectrum

For methanolic extracts and flours.

Other possibility for phenolics determinations….and other determinations

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Use of spectroscopy en the stimation of thecomposition of Tarwi (Including phenolics)

• Construction of IR spectrum in the MIR Region.

• Use of FTIR spectromenter Portable. Thermo

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Courtesy Dr Luis Rodriguez-Saona.

Atenuatted Total Reflectance- Fourier Transform Infrared (ATR- FTIR) spectrometer (Tru Defender TM- Thermo Fisher).

Read the spectrum of Lupin Flour…

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Spectrometer Tru Defender TM-Thermo FisherWhole Lupin Flour

MIR Spectrum characteristic of L. mutabilis flour

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PC1

PC2

PC3

933 1220.270874 1507.393433 1794.515991

Wavenumber

Dis

cri

min

ating

Pow

er

Bitter

Non/bitter

SIMCA ClasificationSoft Independence modeling of class analogy

Fast classification between bitter and debittered tarwi flour.

Fast method phenolics using FT IR spectrum

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0.1 g of defatted flour

0.5 ml of methanol

After centrifugation…

Let evaporate 20 ul extractover ATR sensor

Chemiometricssoftware: multivariatestatistical análisis. Weused Pirouette (v 4.0, Infometrix, USA)

Collect FT IR Spectrum

Ultrasound

FT- IR spectrum for 52 genotypes (methanolic extracts)

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Full Data

2081.638672 3517.251465

Wavenumber

-0.2

0.0

0.2

0.4

Re

sp

on

se

MIR: 4000 – 700 cm -1


Ongoing workfor the fastdeterminationsof phenolicsbased on FT IR spectrum

480 500 520

Measured Phenolic Content

480

490

500

510

520

Pre

dic

ted P

henolic

Conte

nt

4

n= 32; Factors=4; SEC=6.93;

SECV=6.43; R val=0.86

Partial Least Square regression (PLSR) plot for Total Phenolic contentusing MIR region (1837- 1421 cm-1)

Preliminary results for calibration phenolicsextract with FT-IR MIR……..needs improvement

Full Data:PLS:Y1/phenolics:Regression Vector

Wavenumber (cm-1)

-1

0

1

Reg

ressio

n V

ecto

r

4

Phenolics regression vector

1593 1737

Conclusions

• The 52 bitter genotypes explored showed a variability in content ofphenolics of 21.6%. Values ranged between 428 and 526 mg Ac galico/100 g.

• Phenolic content decreased for all ecotypes analyzed after debittering. The type of phenolic that remains after debitteringneeds to be analyzed.

• The FT-IR spectroscopy to be improved would allow thecharacterization of high numbers of material in germoplasm Banks.

• FT-IR spectroscopy confirms important changes in compositionbetween bitter and unbitter siamples that allows a rapid clasificationof lupin flours.

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Aknowledgements

• We acknowledge Dr. Luis Rodriguez-Saona for kindly provide the portable FT-IR (ATR-MIR Thermo) and to the PNIA project for Tarwidirected by Felix Camarena Mayta for funding.

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