The Analysis of Monomeric Anthocyanin by pH Differential Method

Is Not Appropriate for Certain Anthocyanins

Abdullah Muzi Marpaung and Kevin Hanandi Tjahjadi

Food Technology Department, Swiss German University Tangerang, Banten, Indonesia

Keywords: Anthocyanins, Color Intensity, Flavylium Cation, Hemiketal, pH Differential.

Abstract: The light absorbance at pH 1 and 4 of 22 anthocyanin-source plant extracts was studied. Each one gram of

fresh sample macerated in 4 ml 0.1 N HCl-Ethanol 96% (1:9) for an hour, then diluted in buffer solution pH

1 and 4 with various dilution factor. The extract spectrophotometrically scanned at visible region (400 – 700

nm), then the λmax, color intensity, browning index (BI), and violet index (VI) determined. The λmax of

extracts were widely vary from 508 nm to 548 nm. Based on the BI the relatively high color quality at pH 4

exhibited by Clitoria ternatea (CT), Dendrobium sonia (DS), Ipomoea tricolor (IT), Dianella ensifolia (DE)

and Melastoma malabathricum (MM) extract. Based on the VI, CT and DE exhibited bluish-purple color at

pH 4, while DS was redish-purple, IT was purplish-red, and MM was red. Based on the light absorbance, the

extracts might be classified into three types. The A-type exhibited very low intensity of flavylium cation

(AH

) species at pH 4 because of the hydration to colorless hemiketal. The B-type had relatively high intensity

of AH

. The C-type showed the existence of purple and blue quinonoidal base species. The measurement of

monomeric anthocyanin by pH differential method is based on assumption that the anthocyanin colorless at

pH 4.5. Therefore, the method was not suitable for the B- and C-type anthocyanin-source plant extract.

1 INTRODUCTION

Anthocyanins are the largest water-soluble pigment

that produce various color like red, purple, and blue.

There are more than 900 types of anthocyanin found

in plants (Yoshida, et al., 2009). They also provide

beneficial health effects to the human body as an

antioxidant (Gradinaru, et al., 2003; Patras, et al.,

2010), antidiabetic (Belwal, et al., 2017), anticancer

(Patras, et al., 2010) and anti-inflammatory (Lee, et

al., 2017).

The anthocyanin content in a plant extract is

commonly determined spectrophotometrically as

monomeric anthocyanin by the pH-differential

method (Lee, et al., 2005). The analysis based on the

characteristics of monomeric anthocyanin that may

appear as six different species depend on the pH. At

pH 1, anthocyanin exists as the red flavylium cation

species (AH

). Meanwhile, at pH 4.5 the pigment

exists as the colorless hemiketal (B). Hence, the

difference in light absorbance represent the

concentration of the pigment. The polymeric form –

the product of anthocyanin degradation – is resistant

to color change with change of pH and appear as red

both in pH 1 and 4.5 and is not measured by the pH-

differential method.

As the part of our research to find the potential

source of anthocyanins for natural food colorant, we

evaluated the light absorbance of various plant extract

at pH 1 and 4. We found that several anthocyanins

show relatively high color intensity that make them

not suitable to be analyzed by the pH differential

method.

2 MATERIALS AND METHODS

2.1 Materials

Twenty-two samples of anthocyanin-source flowers

and fruits collected from various location in

Indonesia. Seventeen flowers included in this

research were Agapanthes umbellatus, Antirrhinum

majus, Bauhinia purpurea, Clitoria ternatea,

Chrysanthemum x morifolium, Dendrobium sonia,

Dianthus caryophyllus, Gladiolus hortunalus,

Hydrangea macrophilla, Ipomoea tricolor,

Kalanchoe blossfeldiana, Petunia integrifolia,

Marpaung, A. and Tjahjadi, K.

The Analysis of Monomeric Anthocyanin by pH Differential Method is not Appropriate for Certain Anthocyanins.

DOI: 10.5220/0009985400002964

In Proceedings of the 16th ASEAN Food Conference (16th AFC 2019) - Outlook and Opportunities of Food Technology and Culinary for Tourism Industry, pages 26-30

ISBN: 978-989-758-467-1

Ruellia tuberosa, Sinningia speciose, Stachytarpheta

jamaicensis, Tibouchina semidecandra, and Torenia

fournieri. Five fruits included were Clidemia hirta,

Dianella ensifolia, Melastoma malabathricum,

Rhodomyrtus tomentosa, and Vitex pinata.

The hydrochloric acid, ethanol 96%, potassium

chloride, buffer solution pH 4 (citric acid-sodium

hydroxide-hydrogen chloride) were obtained from

Merck®. All reagents were analytical grade and used

without further purification. The buffer pH 1 made

from hydrochloric acid and potassium chloride.

2.2 Maceration and Extract

Preparation

One gram of fresh sample macerated in 4 ml 0.1 N

HCl-Ethanol 96% (1:9) for an hour. The suspension

was filtered through filter paper Whatman 40 (8 µM).

The extract was diluted with buffer solution pH 1 and

4 with various dilution factor (DF) (two to twenty)

depend on the initial intensity of the extract.

2.3 Light Absorbance

The light absorbance at visible region (400 nm - 700

nm) of all extracts was scanned by UV-Vis

spectrophotometer (T60 visible PG Instrument) to

determine the wavelength with maximum absorbance

(λ

max

), relative color intensity (RCI), browning index

(BI) and violet index (VI).

The CI was (A

λmax

– A

700

) x DF (Cisse, et al.,

2012). The RCI was the CI at pH 4 divided by the CI

at pH 1. The BI determined by (A

420

-A

700

)/ (A

λmax

700

) (Cisse, et al., 2012). The VI determined by

580

-A

700

)/(A

520

-A

700

) (Cisse, et al., 2012). A

λmax

was

the absorbance at wavelength with maximum

absorbance, A

420

was absorbance at wavelength 420

nm, A

580

was the absorbance at 580 nm, A

520

was the

absorbance at 520 nm, A

700

was the absorbance at 700

nm for haze correction.

3 RESULTS AND DISCUSSION

3.1 λ

max

, Browning Index, and Violet

Index

All anthocyanin-source extracts showed red color at

pH 1 that represent the presence of flavylium cation

(AH

) species. The λ

max

varied from 508 nm to 548

nm. The shortest λ

max

exhibited by the

Chrysanthemum x morifolium (CM) extract, while the

longest λ

max

belonged to the Clitoria ternatea (CT)

extract (Table 1). The variation was affected by the

type of anthocyanin aglycon (Bueno, et al., 2012), the

number of glycosyl group, acylation and

copigmentation (Gauche, et al., 2010) and the

presence of metal complexation (Yoshida, et al.,

2009). The presence of acyl group tends to increase

the λ

max

. The CT extract, for instance, contains 9 types

of anthocyanin that have two to four acyl groups

(Kazuma, et al., 2003). The TS extract contains

anthocyanin with one acyl group (Lowry, 1976).

Meanwhile, the main anthocyanins in MM extract

have no acyl group (Aishah, et al., 2013). The λ

max

the extracts were 548, 534 and 514 nm, respectively.

Figure 1 depicted the chemical structure change

of simple anthocyanin as the change of pH (Trouillas,

et al., 2016). The red AH

in most anthocyanin-

source extract thermodynamically hydrated to

colorless B as the pH of solution increase to 4 to 5.

As the result, the color intensity of the extract is

dramatically decrease and the λ

max

disappear. Eight of

twenty-two extracts studied exhibited no λmax at pH

4 (Table 1). Six extracts had λ

max

that similar with the

max

at pH 1. The hypsochromic shift (the shift of λ

max

toward a shorter wavelength) occurred in two

extracts. Meanwhile, the bathochromic shift (the shift

of λ

max

toward a longer wavelength) appeared in six

extracts. The wide bathochromic shift occurred in CT,

DS, and DE extracts: 24, 17, and 36 nm, respectively.

The wide shift indicated the kinetic deprotonation of

to form purple quinonidal base A (Trouillas, et

al., 2016).

Browning index (BI) is a common parameter to

measure the color quality of an anthocyanin source

extract (Cisse, et al., 2012). The increase of browning

index indicates the decrease of desirable color (red,

purple or blue) and or the increase of undesirable pale

yellow color (A420) that contributed by the chalcone

species (Reyes & Cisneros-Zevallos, 2007). The

relatively small BI (< 0.5) exhibited by CT, DS, IT,

DE, and MM extracts. The smallest BI belonged to

CT extract.

The other common parameter to determine the

color quality of anthocyanin is violet index (VI) that

measure the ratio of intensity of purple color

(represented by the absorbance at 580 nm) to the

intensity of red color (represented by the absorbance

at 520 nm). Twenty extracts exhibited red color at pH

4, that represented by the relatively low VI (< 1). The

CT, DS, and DE had VI >1 and exhibited purple to

purple blue color.

The Analysis of Monomeric Anthocyanin by pH Differential Method is not Appropriate for Certain Anthocyanins

Table 1: The λ

max

of all anthocyanin-source extract studied at pH 1 and 4 and their color quality at pH 4.

Plant Code λ

max

(nm) Color quality at pH 4

pH 1 pH 4 RCI BI VI

Flower

apanthes umbellatus

U 536 536 0.96 0.91 0.56

ntirrhinum ma

M 530 537 0.85 0.63 0.51

Bauhinia purpurea B

522 522 0.22 1.19 0.59

Clitoria ternatea C

548 572 1.28 0.16 1.94

Chr

santhemum

mori

olium CM 508 - 0.41 1.76 0.48

Dendrobium sonia DS 526 543 0.89 0.42 1.12

Dianthus car

oph

llus D

525 - 0.18 1.67 0.38

Gladiolus hortunalus G

520 - 0.07 2.79 0.48

dran

ea macrophilla

M 525 525 0.15 1.55 0.47

pomoea tricolo

536 539 0.71 0.28 0.49

alanchoe bloss

eldiana

B 520 - 0.50 2.36 0.42

Petunia inte

olia P

531 - 0.43 1.77 0.58

Ruellia tuberosa RT1 526 - 0.11 2.03 0.60

Sinnin

ia speciose SS 520 - 0.07 2.54 0.42

Stach

tarpheta

amaicensis SJ 527 521 0.15 1.62 0.60

Tibouchina semidecandra TS 534 - 0.15 1.94 0.65

Torenia

ournieri TF 532 525 0.30 1.24 0.50

Fruit

Clidemia hirta C

521 526 0.14 0.67 0.35

Dianella ensi

olia DE 532 568 1.17 0.29 1.59

elastoma malabathricum

M 514 513 0.29 0.48 0.19

Rhodom

rtus tomentosa RT2 511 511 0.27 0.61 0.16

Vitex pinata V

516 517 0.35 0.79 0.41

3.2 Classification of

Anthocyanin-source Extract based

on the Light Absorption

The light absorption of the anthocyanin-source

extracts studied might classified into three types as

shown in Figure 2. The A-type represented the most

common anthocyanin that show very low color

intensity at pH 4 as the result of the conversion of red

to B (Lee, et al., 2005). The pale red color in the

extract at pH 4 was the polymeric form of

anthocyanin that is resistant to color change because

of the pH change (Lee, et al., 2005).

At the B-type, the CI of the extracts at pH 4 was

slightly lower than the CI at pH 1. There were three

extracts include in this group: AU, AM, IT. The

retaining color of the three extracts at pH 4 were 96%,

85%, and 71%, respectively. Probably, the relatively

high intensity indicated that the hydration of AH

B was blocked because of the presence of

intramolecular copigmentation. The occurrence of

intramolecular copigmentation involving three acyl

groups and the anthocyanin chromophore in heavenly

blue anthocyanin of IT extract was already

determined (Yoshida, et al., 2009).

An interesting characteristic shown by the C-type

extracts (CT, DS, and DE). At pH 4, all extracts

exhibit two λ

peak

and one λ

shoulder

that represent all the

colored species of anthocyanin: red AH

, purple A,

and blue A

. This unique light absorption profile was

reported as the unique characteristic of anthocyanin

that has acyl group located at the ring B (Baublis, et

al., 1994). The presence of acyl group at the ring B of

anthocyanin was identified in CT and DE extract

(Yoshida, et al., 2009; Kazuma, et al., 2003).

The CI of CT and DE extract at pH 4 was higher

than at pH 1, while the relative CI of DS extract was

0.89. The higher intensity was possible because the

purple and blue species absorb light more intense than

16th AFC 2019 - ASEAN Food Conference

Figure 1: Chemical structures of six simple anthocyanins in aqueous solution (Trouillas, et al., 2016).

Figure. 2: Three different light absorption profile of 22 anthocyanin-source extract studied at pH 1 and 4. The A-type,

consisted of 16 sources, exhibit very low intensity at pH 4. The B-type, consisted of three sources, exhibit a slight lower

intensity at pH 4. The C-type, consisted of three sources, showed two λ

peak

and one λ

shoulder

. The color intensity at pH 4 was

higher than at pH 1.

O-glu

Flavylium cation AH

O-glu

+ H

Hemiketal

O-glu

Quinonoidal bases

cis-Chalcone

trans-Chalcone Ct

O-glu

- H

O-glu

Anionic Quinonoidal bases

0.0

0.2

0.4

0.6

0.8

1.0

400 500 600 700

Absorbance (AU)

Wavelength (nm)

pH1

pH 4

0.0

0.2

0.4

0.6

0.8

1.0

400 500 600 700

Absorbance (AU)

Wavelength (nm)

pH1

pH 4

0.0

0.2

0.4

0.6

0.8

1.0

400 500 600 700

Absorbance (AU)

Wavelength (nm)

pH1

pH 4

The Analysis of Monomeric Anthocyanin by pH Differential Method is not Appropriate for Certain Anthocyanins

the red species (Yoshida, et al., 2009). 4, that

represented by the relatively low VI (< 1). The CT,

DS, and DE had VI >1 and exhibited purple to purple

blue color

3.3 Determination of Monomeric

Anthocyanin

The determination of monomeric anthocyanin by pH

differential method is a rapid method that widely

accepted to determine the anthocyanin content in a

plant extract or juice (Lee, et al., 2005). In the

method, an assumption made that the monomeric

anthocyanins exhibit little or no light absorbance at

pH 4.5. Meanwhile, the polymeric anthocyanins will

absorb at the pH.

As demonstrated in Figure 2, the A-type of

anthocyanin-source extracts we studied fit the

assumption. Hence, the monomeric anthocyanin

might appropriately be determined. However, the B-

type and C-type exhibit relatively high light

absorption at pH 4 that probably because they contain

polyacylated anthocyanins. Consequently, the use of

pH differential method to determine the monomeric

anthocyanin content in B-type and C-type

anthocyanin-source extract was not suitable.

4 CONCLUSIONS

The twenty-two anthocyanin-source plant extract

exhibit different light absorption at pH 4 that can be

classified into three types. The A-type exhibited very

low light absorption of flavylium cation (AH

)

species, the B-type showed relatively high intensity

of AH

, while in the C-type the significant amount of

purple quinonoidal base (A) and blue anionic

quinonoidal base (A

) observed. Therefore, the use of

pH differential method to determine the monomeric

anthocyanin content was not appropriate to be applied

to B- and C-type of anthocyanin source extract.

The spectrophotometric scan at visible light

region, both at pH 1 and 4, of an unidentified

anthocyanin-source plant extract is suggested before

the examination of pH differential method.

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