035 Jose L. Paredes S-Phytochemical Screening and Quantification of Total Flavonoids of Canned

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The consumption of canned asparagus all over the world

has considerably increased over the recent years. In 2004, 45%

of the total worldwide production of asparagus was canned,

1. INTRODUCTION

PHYTOCHEMICAL SCREENING AND QUANTIFICATION

OF TOTAL FLAVONOIDS OF CANNED WHITE

ASPARAGUS “ASPARAGUS OFFICINALIS L.” I N T E R N A T I O N A L

.countries for this production. MTC International is one of the

world’s largest companies with 14 years of experience trading

canned asparagus from China and Peru, with huge distribution

in the big European market. Based in Germany, the strength of

MTC International is its group of professional Peruvianengineers who are in charge of the control in production and

quality assurance.



Considering the importance of canned asparagus in the

market, MTC International is also paying attention on scientific



ASPARAGUS “ASPARAGUS OFFICINALIS L.”

INTRODUCTION

I N T E R N A T I O N A L

researches about asparagus. For that reason, this presentation

is to explain you about the phytochemical screening of samples

of canned white asparagus in order to know the composition of

secondary metabolites and quantification of flavonoids.



SECONDARY METABOLITES




Secondary Metabolites are chemical compounds

(flavonoids, alkaloids, terpenoids, steroids, quinones, saponins,


distribution than primary metabolites. They are not

indispensable for the plant that contains them; at least their

metabolic functions have not been discovered yet. They are not

as important as the primary metabolites (glucids, proteins and

amino acids) which are widely distributed and indispensable inthe normal growth, development, or reproduction of the plant.






Flavonoids are polyphenolic compounds with high capacity

of inhibition of free radicals in human beings which derivates from

stress, antibiotic consumption, pollution, etc. This is called

FLAVONOIDS


would imply a degradation or aging of the cells in human organism.

The flavonoids have aroused considerable interest recently

because of their potential beneficial effects on human health. They

have been reported to have antiviral, anti-allergic, anti-platelet,

anti-inflammatory and antitumor activities.A researching of 2002 in the Netherlands said that the

consumption of flavonoids per person was 23mg per day, which

70% was quercetin (Ochoa, C. 2004).



2. MATERIALS AND Y METHOD

The phytochemical screening of canned asparagus was

done using the methodology described by Martinez and




, . , , .The spectrophotometric method to quantify the total

number of flavonoids expressed as quercetin is the one

described by Kostennikova Z1 (Gutierrez, Y.I. 2000).

The analyzed product is white peeled asparagus from the

variety “UC-157F2” sterilized in glass jar 370ml and 16cm at117oC with 17 minutes.



Preparation of the sample




Sample preparation (screening) Sample preparation



2.1. Alkaloids Identification5 gr of sample, canned

white asparagus

Extraction /5 min /waterbath

HCl at 5%




Filtration

Mayer Draggendorf Wagner Hager

Tests

If turbidity or precipitates are observed in at least three tubes, it is considered that

the sample contains alkaloids

Solid






Alkaloids test






Alkaloids test (not found)



2.2. Flavonoids and Leucoanthocyanidins5 g. of sample, canned

white asparagus

Extraction

Ethanol

Water bath/ 5 min




Filtration

Precipitation

Filter

Flavonoids test Leucoanthocyanidins test

Solid

Plumbic Acetate at 4 % / Acetic Acid at 0.5 %.

Rest 15 min

Solid

Magnesium filings+ HCl cc. 1ml of HCl cc/boil for 15 min






Flavonoids


Leucoanthocyanidins

Test






Test tubes with: magnesium filings, 2ml of filtered product and several drops of HCl

cc. If colors like orange, pink, red, or purple appear, it is a proof of the existence of

flavonoids in the sample.



The color is lightly pink,




moderated quantity of

leucoanthocyinidins



2.3. Saponins, Tannins and Quinones10 g of sample, canned white

asparagus

Extraction

Ethanol/water bath 10 min.




Filtration

Liquid-Liquid Extraction

Organic phase Aqueous phase

Filtration

Tannins Saponins Quinones

Solid

Separation Funnel

2 portions of Dichloromethane

20 ml. each

Solid



Tannins

Centrifugation

Saponins

Shake

Quinones

Heating (waterbath)supernatant

4 ml of filtrate

1 ml of filtrate

+ 1ml of gelatina-salt

5 ml of filtrate

1 ml of H2O2 at 20%

1 ml of H2SO4 at 50%

5 ml of toluene




Redissolution

L-L Extraction

Toluenephase

Shake

Aqueousphase

, . ,2 drops of FeCl3 at 10%

The presence of green, blue

or black color is a proof of

the presence of tannins

The presence of

froth is a proof of

the presence of

saponins

1 ml of NaOH at 5%

The presence of red

color is a proof of

the presence of

Quinones






Saponins and Tannins Test






Saponins test (moderate)






no presenta

taninos

Tannins

are not

present






Separación de

fases.

Phases

separationQuinones test (low)



10 g. of sample, canned whiteasparagus

Heating

200 ml of H2O

Boiling/5 min

2.4. Recognition of Anthocyanins




Filtering

Color of ReactionNo 1

Color of ReactionNo 2

Solid

2 ml of filtrate +

1 ml of NaOH

2 ml of filtrate +

1ml of HCl

If the colors of both reactions are different, it is

a proof of the presence of Anthocyanins






Anthocyanins test (not present)



2.5. Quantification of Total Flavonoids6,25 g of sample,canned white

asparagus

Re-flowing (2 h at90ºC)

Solid20 ml of H2SO4 +

20 ml of Ethanol at 50%Wash with 40 ml H20

(10-15oC)

70 ml of Ethanol at 50oC




Filtering

Evaporate

Cooling down

Filtering

Dissolve

Filling

Solid

Water bath/half of

the initial volume

Ice bath/30 min

Total 100 ml

To make a scan in the

spectrophotometer

filtrate






Samples for quantification of

total flavonoids

Quantification of total flavonoids

Sample in Sulfuric

Acid at 10% and

Ethanol at 50%






Re-flowing equipment (Quantification of total flavonoids)






Quantification of total flavonoids

(Filtration)

Obtaining of flavonoids in paper filters






Addition of Ethanol at 50oC to the

flavonoids in paper filters

Samples to be scanned in

spectrophotometer at 258nm



2.6 Calibration Curve

• Check 0,002 g of quercetin and dissolve with

ethanol at 96 % till com lete 10 ml.




• Prepare following: 1,11µg/ml; 3,33µg/ml;

5µg/ml; 10µg/ml.

• Then read the absorbances of each solution in

the spectrophotometer UV-VIS at 258 nm.



y = 0,1029x + 0,002

R² = 0,99950.8

1

1.2

c e

Calibration Curve using Quercetin as blank




0

0.2

0.4

0.6

0 2 4 6 8 10 12

A b s o r b a n

Quercetin concentration (µg/mL)

Absorbancia Lineal (Absorbancia)






Quercitin Preparation of the pattern curve with quercitin

Quartz cubes for spectrophotometer Spectrophotometer UV






Preparation of quercitin solutions for the

pattern curve

Spectrophotometric scanning of the samples



Secondary Metabolites Found Quantity

Alkaloids -

3.1. Phytochemical Analysis3. RESULTS AND DISCUSSION




Flavonoids +++++

Leucoanthocyanidins +++

Saponins +++

Quinones ++

Tannins -Anthocyanidins -

Excelent: +++++ ; Good: ++++; Moderate: +++; Low:++; Not present :-



It is necessary to emphasize the importance of the presence

of flavonoids in canned white asparagus because of its great

antioxidant properties. Among all, the most valuable biological




3.2. Discussion


.These advantages have been the subject for countless

researches, mainly in clinical and nutritional matters.

Pharmacological doses of dietary antioxidants, commonly

recommended in all over the world, such as combinations of

vitamins E plus C and β-carotene, do not produce the desired effects

or sometimes these are harmful to consumer’s health. Therefore, toget a better antioxidant action it is preferred the use of a mixture of

flavonoids and tannins in a daily diet(Perez, 2003)



3.3. Quantification of Total Flavonoids

The white canned asparagus processed in Trujillo contains

1.16 mg of equivalent quercetin per gram of dry sample, which

expressed in drained weight contains 9.344 mg of equivalent

quercetin per 100 grams of drained weight.

These results are similar of those got by Fuentes




(Fuentes, 2009) who found from 0.6 to 1.8 mg of equivalentquercetin per gram of dry asparagus fiber. According to Lock

(Lock, 2006), celery contains 0.0412 mg of equivalent quercetin per

gram of dry sample, which compared with canned asparagus it is

still low.

Researches on different varieties of artichokes

(Curadi, 2005) report the content of total phenols (expressed as

equivalent catechin per gram of dry weight) higher than those in

canned asparagus, but we have to consider that artichokes are fresh

and asparagus are treated at high temperatures where flavonoids



4. CONCLUSIONS

Canned white asparagus "Asparagus officinalis" from variety

UC-157F2, sterilized at 117⁰C and 17 minutes of holding

process, have the following secondary metabolites in their chemical




compos on: avono s n re a ve y g amoun s, a mo era eamount of leucoanthocyanidins, a moderate amount of saponins

and none presence of quinones.

The analyzed canned asparagus have 1.16 mg of total

flavonoids expressed as equivalent quercetin per one gram of dry

sample or 9.34 mg of equivalent quercetin per 100 g of drained

sample, and the content of flavonoids in canned white asparagus

exceeds the one in some vegetables such as celery and artichokes.

035 Jose L. Paredes S-Phytochemical Screening and Quantification of Total Flavonoids of Canned

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