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.

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Considering the importance of canned asparagus in the

market, MTC International is also paying attention on scientific

PHYTOCHEMICAL SCREENING AND QUANTIFICATION

OF TOTAL FLAVONOIDS OF CANNED WHITE

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.

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SECONDARY METABOLITES

PHYTOCHEMICAL SCREENING AND QUANTIFICATION

OF TOTAL FLAVONOIDS OF CANNED WHITE

ASPARAGUS “ASPARAGUS OFFICINALIS L.” 

Secondary Metabolites are chemical compounds

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

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

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.

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PHYTOCHEMICAL SCREENING AND QUANTIFICATION

OF TOTAL FLAVONOIDS OF CANNED WHITE

ASPARAGUS “ASPARAGUS OFFICINALIS L.” 

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

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

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).

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2. MATERIALS AND Y METHOD

The phytochemical screening of canned asparagus was

done using the methodology described by Martinez and

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

, . , , .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.

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Preparation of the sample

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

Sample preparation (screening) Sample preparation

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2.1. Alkaloids Identification5 gr of sample, canned

white asparagus

Extraction /5 min /waterbath

HCl at 5%

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

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

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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

Alkaloids test

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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

Alkaloids test (not found)

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2.2. Flavonoids and Leucoanthocyanidins5 g. of sample, canned

white asparagus

Extraction

Ethanol

Water bath/ 5 min

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

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

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PHYTOCHEMICAL SCREENING AND QUANTIFICATION

OF TOTAL FLAVONOIDS OF CANNED WHITE

ASPARAGUS “ASPARAGUS OFFICINALIS L.” 

Flavonoids

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

Leucoanthocyanidins

Test

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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

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.

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The color is lightly pink,

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

moderated quantity of 

leucoanthocyinidins

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

asparagus

Extraction

Ethanol/water bath 10 min.

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

Filtration

Liquid-Liquid Extraction

Organic phase Aqueous phase

Filtration

Tannins Saponins Quinones

Solid

Separation Funnel

2 portions of Dichloromethane

20 ml. each

Solid

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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

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

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

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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

Saponins and Tannins Test

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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

Saponins test (moderate)

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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

no presenta

taninos

Tannins

are not

present

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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

Separación de

fases.

Phases

separationQuinones test (low)

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10 g. of sample, canned whiteasparagus

Heating

200 ml of H2O

Boiling/5 min

2.4. Recognition of Anthocyanins

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

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

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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

Anthocyanins test (not present)

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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

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

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

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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

Samples for quantification of 

total flavonoids

Quantification of total flavonoids

Sample in Sulfuric

Acid at 10% and

Ethanol at 50%

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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

Re-flowing equipment (Quantification of total flavonoids)

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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

Quantification of total flavonoids

(Filtration)

Obtaining of flavonoids in paper filters

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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

Addition of Ethanol at 50oC to the

flavonoids in paper filters

Samples to be scanned in

spectrophotometer at 258nm

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2.6 Calibration Curve

• Check 0,002 g of quercetin and dissolve with

ethanol at 96 % till com lete 10 ml.

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

• 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.

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y = 0,1029x + 0,002

R² = 0,99950.8

1

1.2

     c     e

Calibration Curve using Quercetin as blank

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

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)

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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

Quercitin Preparation of the pattern curve with quercitin

Quartz cubes for spectrophotometer Spectrophotometer UV

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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

Preparation of quercitin solutions for the

pattern curve

Spectrophotometric scanning of the samples

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Secondary Metabolites Found Quantity

Alkaloids -

3.1. Phytochemical Analysis3. RESULTS AND DISCUSSION

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

Flavonoids +++++

Leucoanthocyanidins +++

Saponins +++

Quinones ++

Tannins -Anthocyanidins -

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

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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

PHYTOCHEMICAL SCREENING AND QUANTIFICATION

OF TOTAL FLAVONOIDS OF CANNED WHITE

ASPARAGUS “ASPARAGUS OFFICINALIS L.” 

3.2. Discussion

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

.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)

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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

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

(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

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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

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

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.