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DualfunctionsofCuNO32asantifungalandcolourstabilizerforcoatingpaintfilmconsistingofchlorophyll

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Dual functions of Cu(NO3)2 as antifungal and colour stabilizer for coating

paint ﬁlm consisting of chlorophyll

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Pigment and Resin Technology · March 2019

DOI: 10.1108/PRT-11-2017-0086

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Dual functions of Cu(NO

)

2

as antifungal and colour stabilizer for coating paint film consisting of chlorophyll

L. Sulaiman, Z.H.Z. Abidin, S.A.M. Yunus, N.A. Mazni, A.N. Hadi, A.K. Arof, K. Simarani,

Article information:

To cite this document:

L. Sulaiman, Z.H.Z. Abidin, S.A.M. Yunus, N.A. Mazni, A.N. Hadi, A.K. Arof, K. Simarani, (2019) "Dual functions of Cu(NO3)2

as antifungal and colour stabilizer for coating paint film consisting of chlorophyll", Pigment & Resin Technology,

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Dual functions of Cu(NO

)

as antifungal and

colour stabilizer for coating paint

ﬁlm

consisting of chlorophyll

L. Sulaiman, Z.H.Z. Abidin, S.A.M. Yunus, N.A. Mazni, A.N. Hadi

and

A.K. Arof

Centre for Ionics University of Malaya, Department of Physics, Faculty of Science, University of Malaya, Kuala Lumpur, Malaysia, and

K. Simarani

Institute of Biological Sciences, Faculty of Science, University of Malaya, Kuala Lumpur, Malaysia

Abstract

Purpose

– This paper aims to discuss about the beneﬁts of Cu(NO

)

2

applied in coating paint

ﬁlm that consists of plant extract from Cassia Alata.

Cu(NO

3

)

has been added to the coating paint

ﬁlm as a colour stabilizer and as an antifungal property by forming a zone of inhibition towards

fungal growth.

Design/methodology/approach

– The coating paint ﬁlm resin consists of a mixture of poly (methyl methacrylate) (PMMA) and acrylic polyol. The

coating paint

ﬁlm’s performances were observed in terms of visible optical and physical appearance.

Findings

– When exposed to the fungus Trichoderma Sp., a zone of inhibition of 208 mm

was formed around the coating paint

ﬁlm and therefore it

gave the coating paint

ﬁlms an antifungal property. The commission internationale de l’éclairage (CIE) L*a*b* colour coordinate system was used

to identify the colour changes in the coating paint

ﬁlm and it was found that the addition of Cu(NO

)

to the coating paint

ﬁlm has a better colour

stability, as it has a lower standard deviation (STD) value of 6.46 than the pure dye coating paint

ﬁlm of 8.95. The gloss of coating paint ﬁlm with

the addition of Cu(NO

)

2

was found to be have a more stable gloss based on its smaller STD value at both angles taken at 20° and 60°.

Research limitations/implications

– The Cassia Alata plant has different amount of antifungal properties based on location its growth.

Practical implications

– The coating paint ﬁlm developed in this study is suitable for indoor applications.

Originality/value

– The plant extract from the Cassia Alata gives the coating paint ﬁlm an antifungal property.

Keywords Antifungal, Copper (II) Nitrate, Coating paint

ﬁlms, Cassia Alata, Trichoderma Sp.

Paper type Research paper

Introduction

The main material used in this project is a natural green dye

that consists of chlorophyll extracted from plants. This dye has

been used mainly as a traditional fabrics dye and medicine in

the early twentieth century. The source of plant used is Cassia

Alata that is widely grown in South East Asia countries. The

plant extract is known to show a range of antifungal activity

against fungi, bacteria and many other microorganisms

(

Ibrahim and Osman, 1995

;

Khan et al., 2001

;

Somchit et al.,

2003

). The fungus used in this experiment is Trichoderma Sp.

that grows rapidly in room temperature condition, reproduced

by releasing conidial spores into the air and commonly found in

soil, decaying wood, food, etc. It has the capacity of producing

an enzyme called cellulase that decays cellulose (

łaszczyk

et al., 2014

;

opez-Bucio et al., 2015

The coating paint

ﬁlm consists mainly of PMMA and acrylic-

polyol polymer. The poly (methyl methacrylate) (PMMA) is a

synthetic resin produced from the polymerization of methyl

methacrylate that is a type of transparent and rigid plastic. The

glassy PMMA has an improved light scattering and absorption in

the 490-680 nm range (

Christ and Marhic, 1982

), which is

suitable for the colour given out by the green dye pigment. It has

environmental stability, which is better than most plastics such as

polystyrene, and is therefore, often chosen as a material for

outdoor applications. However, PMMA has a low adhesion

characteristic, which causes it to be dif

ﬁcult to be applied as resin

in coating paint

ﬁlm. Therefore, acrylic polyol is mixed with

PMMA to improve its adhesion characteristic. The acrylic polyol

is used as a coating resin due to its outstanding properties, such as

its transparency and colour stability. It is also great for dispersing

pigments and is generally unreactive, which makes it suitable to

be mixed with pigments, as it is stable.

Copper (II) nitrate, also known as copper dinitrate or cupric

nitrate with molecular formula of Cu(NO

)

, is a bluish-green,

odourless crystalline material. In this research, it has been used as

an additive for colour stability in the chlorophyll dye to preserve

its green colour, as chlorophyll starts to oxidize after a period of

time and also acts as an antifungal agent. We used copper (II)

The current issue and full text archive of this journal is available on

Emerald Insight at: www.emeraldinsight.com/0369-9420.htm

Pigment & Resin Technology

[DOI

10.1108/PRT-11-2017-0086

]

The authors would like to express gratitude to the University of Malaya for

providing the research grant (RP025D-14AFR).

Received 2 November 2017

Revised 12 March 2018

Accepted 30 April 2018

Downloaded by University of Malaya At 22:38 12 March 2019 (PT)

nitrate because copper does not easily oxidize, and is fairly

unreactive.

Chlorophyll

’s molecule structure consists of

prophyrin ring, which functions as light adsorption with a

magnesium ion in the centre and a long hydrocarbon tail that

keeps

the

chlorophyll

embedded

the

photosynthetic

membrane (

Karp, 2009

). When Cu(NO

)

2

is added, it turns

chlorophyll into metallochlorophylls where it replaces the

magnesium in the prophyrin ring with the transition element,

copper. Copper stabilizes the chlorophyll

’s structure due to the

presence of the three-dimensional orbitals in the copper, as this

increases the covalent character of the linkage between the ligand

and the copper acceptor. Therefore, with the copper ion, the

chlorophyll molecule will be more stable and maintain its

structure (

Bechtold and Mussak, 2009

). Besides that, it is well

known that copper has an antifungal property as it has inhibitory

property towards spore germination that is responsible for the

reproduction of fungus. This causes it neither to mature nor to

reproduce and therefore it is known to inhibit the fungal growth.

It is mostly used in pesticide (

Richardson, 1997

). In this work, we

are attempting to develop an antifungal coating paint

ﬁlm that

can maintain its green colour from its natural dye pigment,

chlorophyll, when exposed to the fungus, Trichoderma Sp. with

the help of copper (II) nitrate.

Usop et al. (2016)

developed a coating paint

ﬁlm that

consists of a green dye chlorophyll as colourant. They found

that the addition of Cu(NO

)

to the coating paint

ﬁlm was

able to improve the colour stability after the exposure to UV-A.

The colour degradation (photodegradation) process was

measured using the commission internationale de l

’éclairage

(CIE) L*, a* and b* colour coordinate method. In this

research, the same type of coating paint

ﬁlm was prepared and

exposed to a fungus, Trichoderma Sp., to observe its anti-fungal

characteristic. It is expected that the coating paint

ﬁlm with its

anti-fungus characteristic shows a zone of inhibition against the

fungal growth and has good colour stability. Any formation of

fungus growth nearby or on the coating paint

ﬁlms surface

area causes a biodeterioration process, which leads to

discolouration.

Experimental

The procedure contains the sample preparation of the dye, the

coating paint

ﬁlm sample and the fungus sample.

Dye preparation

For the preparation of the chlorophyll dye, the Cassia Alata

leaves were dried in an enclosed room at room temperature of

27°C for 24 h. Once the leaves were dried, they were cut into

smaller pieces and immersed in absolute ethanol overnight.

The mixture was

ﬁltered and the chlorophyll dye was obtained.

Two dyes were prepared for this experiment, one containing

only the chlorophyll and the other containing chlorophyll and

3 Wt.% copper (II) nitrate, Cu(NO

)

as an additive for colour

stability and to improve the antifungal property.

Preparation of coating paint

ﬁlm sample

The binder was prepared by adding PMMA (MW: 350,000 g

mol

1

) to xylene with a ratio of 3:7 (PMMA: Xylene) by

weight. The mixture was heated in a water bath until it reaches

a temperature of 65°C and stirred overnight with a magnetic

stirrer. Acrylic polyol was mixed into the mixture by using a

glass rod until it fully dissolved with a ratio of 2:8 (Acrylic

Polyol: PMMA

1 xylene) by weight to prepare the binder. To

prepare the coating sample, the binder and dye were mixed

with a ratio 9:1 by volume and was stirred with a glass rod until

it was evenly mixed and was left aside for an hour. Next, a layer

of the mixture was applied on one side of the microscope glass

slide by using a paintbrush and was left to dry for 24 h at room

temperature of 27°C in a dark and dry place. The coating paint

ﬁlm sample was coded as chlorophyll (CHR) and chlorophyll 1

Cu(NO

)

(CHRCU).

Preparation of fungus sample

The spores of the Trichoderma Sp. fungi were extracted by

adding distilled water onto the cultured plate and rubbed

with a sterile loop. The spore suspension was then carefully

transferred into a sterile bottle with 200 ml distilled water and

shaken until it was evenly mixed. The number of spores was

calculated by using a haemocytometer and recorded. Of note,

1 ml of spores was placed on the potato dextrox agar (PDA)

and evenly distributed by using a glass rod spreader until it is

dry. The microscope slide with coating paint

ﬁlm was placed

facing upwards on the fungus sample and sealed with a

para

ﬁlm to avoid contamination.

Characterizations

Colour meter measurement

The CIE L*a*b* colour measurement was used in this

experiment to obtain the colour coordinates of the coating

sample using the Commission Internationale de l

Eclairage

(CIE) 1976 space system or CIE L*a*b*. The colour space

coordinates analysis was conducted by using the

ﬁbre optic

Figure 1 CIE L*a*b* colour coordinates

Figure 2 Determination of zone of inhibition

Dual functions of Cu(NO

)

2

L. Sulaiman et al.

Pigment & Resin Technology

Downloaded by University of Malaya At 22:38 12 March 2019 (PT)

spectrometer with Avasoft (version 7.7) software.

Figure 1

shows the colour coordinates that represent the colour of

L*a*b*. The L*, a* and b* were used to describe the lightness,

redness and yellowness of the colours, respectively. The total

colour difference E was calculated from the CIE L*a*b*

formula as shown below:

Lab

ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ

ð

Þ

1 DaÞ

1 Db

ð Þ

Gloss measurement of the coating sample

The gloss of the coating samples was taken using a KSJ

Instruments MG268-F2 Glossmeter at angles 20

and 60

Gloss is used to measure the amount of light re

ﬂected on the

surface of the substrate. It is an important parameter to

describe the visual appearance of an object. The purpose of

this test is to determine whether the exposure of fungus can

cause alteration to the surface of the coating paint

ﬁlm, as

any changes to the coating paint

ﬁlms surface morphology

can alter the glossiness of the coating paint

ﬁlm sample.

Figure 4 The L*a*b* value of the sample of chlorophyll and copper (II)

nitrate against days

–20

–

9 12 15 18 21 24 27 30

Colour Space Coordinate

Time (Day)

L*a*b* Chlorophyll + Cu(NO

3

)

2

Figure 5 The DE and its derivative of the chlorophyll sample from its

L*a*b* data

9 12 15 18 21 24 27 30

Derivative__ΔE__Time_(Day)__ΔE__its_derivative__of_Chlorophyll_only'>Derivative

ΔE

Time (Day)

ΔE & it's derivative

of Chlorophyll only

Derivative

STD Value : 8.95

Figure 6 The DE and its derivative of the chlorophyll and copper (II)

nitrate sample L*a*b* data

–1

9 12 15 18 21 24 27 30

Derivative

ΔE

Time (Day)

ΔE & it's derivative

of Chlorophyll + Cu(NO

3

)

2

Derivative

STD Value : 6.46

Figure 3 The L*a*b* of the chlorophyll sample against days

–

–20

100

9 12 15 18 21 24 27 30

Colour Space Coordinate

L*a*b* of Chlorophyll only

Figure 7 The graph of gloss of chlorophyll only at different angles (20

and 60

)

120

160

200

9 12 15 18 21 24 27 30

Gloss (GU)

Gloss of Chlorophyll only

20°

60°

Table I Standard division (STD) value of gloss for the CHR and CHRCU

coating sample

Angle (

)

CHR

CHRCU

18.05

5.69

16.40

3.72

Dual functions of Cu(NO

)

2

L. Sulaiman et al.

Pigment & Resin Technology

Downloaded by University of Malaya At 22:38 12 March 2019 (PT)

Zone of inhibition

The zone around the coating paint

ﬁlm sample that has no

visible fungal growth is referred to as a zone of inhibition. It will

either stop the growth or kill the microorganisms and this shows

an antibiotic resistance towards the microorganisms. The zone

of inhibition, which can be seen in

Figure 2

is measured by

using the formula:

Zone of Inhibition

¼ Area of Sample 1 Zone of Inhibition

Þ

– Area of Sample

Results and discussion

Coating paint

ﬁlm sample preparation

The ratio chosen to make the coating paint

ﬁlm sample is

the optimum

–mixing ratio, as it gives a nice colour

distribution, and does not develop bubbles compared to the

other ratios.

Colour meter measurement

The coating paint

ﬁlm sample for both chlorophyll CHR and

chlorophyll

1 Cu(NO

)

CHRCU was exposed to the

fungus Trichoderma Sp. for 30 days. The visual appearance

was measured by using a CIE spectroscopy to measure its

colour coordinates L*a*b*, which are shown in

Figures 3

and

. The CIE L*a*b* parameters were used to identify the

colour difference of the coating paint

ﬁlm samples after

exposure to the fungus. The results obtained show that a*

coordinates for both samples remain constant. The b*

coordinates for CHR coating paint

ﬁlm sample show more

changes than for CHRCU coating paint

ﬁlm sample. This

shows that the CHR coating paint

ﬁlm sample changes

colour from green to brown, whereas the CHRCU coating

paint

ﬁlm sample remains the same after being exposed to

the fungus. The CHRCU coating paint

ﬁlm sample remains

the same colour due to the copper (II) nitrate that acts as a

colour stabilizer.

The

DE for the CHR and CHRCU shows the colour

difference, which is shown in

Figures 5

and

, respectively,

for the 30-day exposure to fungus. It is found that the

standard deviation (STD) value for the value

DE for

CHRCU coating paint

ﬁlm sample is lower than the CHR

coating paint

ﬁlm sample with value 6.46 and 8.95,

respectively. This shows that the colour of the CHRCU

coating paint

ﬁlm sample is much more stable than the CHR

coating paint

ﬁlm sample. This is because the coating paint

ﬁlm sample with the STD value closer to zero has higher

colour stability. The derivative represents the amount of

changes of the

DE coating paint ﬁlm sample within three

days.

The change in colour from green to yellow in the CHR

coating paint

ﬁlm sample is due to the chlorophyll

breakdown in the dye. This degradation in chlorophyll

occurs in plants during the process called senescent, where

the transformation of the green chloroplast into gerontoplast

occurs. Gerontoplast plays an important role in breakdown

of chloroplast protein and recycling nutrients within the

plant (

Kräutler, 2000

). There are other coloured pigments

Figure 8 The graph of gloss of chlorophyll 1 Cu(NO

)

at different

angles (20

and 60

)

120

160

200

9 12 15 18 21 24 27 30

Gloss (GU)

Time (Day)

Gloss of Chlorophyll + Cu(NO

3

)

2

20º

60º

Figure 9 Visible appearance of the effect of fungus on the CHR and CHRCU coating paint ﬁlm sample

Dual functions of Cu(NO

)

L. Sulaiman et al.

Pigment & Resin Technology

Downloaded by University of Malaya At 22:38 12 March 2019 (PT)

in the plant extract besides chlorophyll, such as the

xanthophyll (yellow) and carotene (orange) but because

there are abundant amount of chlorophyll in the plant

extract, it masks the other coloured pigments. Chlorophyll

breaks down faster than the other pigments in the plant and

this causes the colour of the other pigments to be more

visible in the dye extract (

Starr et al., 2011

Gloss measurement

The gloss of the coating surface for both CHR and CHRCU

was measured by using a glossmeter at speci

ﬁc angles (20

and

), as shown in

Figures 7

and

. It is shown that the values for

both samples remain almost constant throughout the 30-day

exposure to the fungus. The STD value for both CHR and

CHRCU can be seen in

Table I

. From the results given, it is

found that the CHR coating sample has a higher gloss value

and also a larger STD value compared to the CHRCU coating

paint

ﬁlm sample. This shows that the CHRCU coating paint

ﬁlm sample has a more stable gloss than the CHR coating

paint

ﬁlm sample has based on its STD value. As there are no

spores on the surface of the coating paint

ﬁlm, it does not affect

the value of its glossiness.

Zone of inhibition

Figure 9

shows the visible appearance of the CHR and

CHRCU coating paint

ﬁlm exposed to the fungus Trichoderma

Sp.. The CHRCU coating paint

ﬁlm can be seen with a clear

zone of inhibition formed around it, whereas in the CHR

coating paint

ﬁlm, there is no clear zone of inhibition formed

around it but we have found that the fungus does not grow on it

and therefore it can be considered as inhibitory. Therefore,

both the coating paint

ﬁlms are considered as a microbistats

that function to prevent the growth of microorganisms and its

spores on its surface rather than kill the microorganisms

(

Koleske, 1995

). It is found that the coating paint

ﬁlm sample

with only Cu(NO

)

has a clear zone of inhibition that can be

seen in

Figure 10

. The following are the speci

ﬁcations of the

samples:

Glass Slide

: 26 76 mm

Zone of inhibition

1 Sample ¼ 2184mm

Zone of Inhibition

¼ 208mm

Magnification

: 30x

Conclusion

Based on the results obtained, the colour measurement of

the chlorophyll coating paint

ﬁlm sample (CHR) changes

from yellowish-green to light brown. This could be due to

the breakdown of chlorophyll, which causes the loss of green

colour in the coating paint

ﬁlm sample (

Matile et al., 1999

The colour of the chlorophyll

1 Cu(NO

)

coating paint

ﬁlm sample remains the same. Besides that, the glossiness of

the CHRCU coating paint

ﬁlm is found to be more stable

than that of the CHR coating paint

ﬁlm. Meanwhile, the

fungus Trichoderma Sp. does not show any signi

ﬁcant

changes on the colour and the surface morphology of the

coating paint

ﬁlm because of the antifungal property in the

coating paint

ﬁlm. Hence, it can be concluded that the Cu

(NO

3

)

acts as an enhancer to the antifungal property of the

plant extract of Cassia Alata and also as a colour stability to

the coating paint

ﬁlm. For further studies, a different type of

fungus or bacterium can be exposed to the coating paint

ﬁlm.

References

Bechtold, T. and Mussak, R. (2009), Handbook of

Natural Colorants, John Wiley & Sons, Chichester,

pp. 249-250.

łaszczyk, L., Siwulski, M., Sobieralski, K., Lisiecka, J. and

ędryczka, M. (2014), “Trichoderma spp. – application and

prospects for use in organic farming and industry

”, Journal of

Plant Protection Research, Vol. 54 No. 4.

Christ, B. and Marhic, M. (1982),

“Light scattering and

absorption

glassy

poly

(methyl

methacrylate)

(PMMA) and polystyrene (PS)

”, Proceedings of SPIE –

The International Society for Optical Engineering, Vol. 297,

pp. 169-172.

Figure 10 Zone of inhibition of Cu(NO

)

2

Dual functions of Cu(NO

)

L. Sulaiman et al.

Pigment & Resin Technology

Downloaded by University of Malaya At 22:38 12 March 2019 (PT)

Ibrahim, D. and Osman, H. (1995),

“Antimicrobial activity of

Cassia Alata from Malaysia

”, Journal of Ethnopharmacology,

Vol. 45 No. 3, pp. 151-156.

Karp, G. (2009), Cell and Molecular Biology: Concepts and

Experiments, 6th ed., John Wiley & Sons, Hoboken, NJ,

pp. 206-212.

Khan,

M.,

Kihara,

and

Omoloso,

(2001),

“Antimicrobial activity of Cassia Alata”, Fitoterapia, Vol. 72

No. 5, pp. 561-564.

Koleske, J. (1995), Paint and Coating Testing Manual, 14th ed.,

ASTM Publication, Philadelphia, PA.

Kräutler,

(2000),

“Chlorophyll breakdown and

chlorophyll catabolites

”, in Kadish, K., Smith, K. and

Guilard, R. (Eds), The Porphyrin Handbook: Chlorophylls

and Bilins: Biosynthesis, Synthesis, and Degradation, 13th

ed., Elsevier, Oxford.

opez-Bucio, J., Pelagio-Flores, R. and Herrera-Estrella, A.

(2015),

“Trichoderma as biostimulant: exploiting the

multilevel properties of a plant bene

ﬁcial fungus”, Scientia

Horticulturae, Vol. 196, pp. 109-123.

Matile, P., Hörtensteiner, S. and Thomas, H. (1999),

“chlorophyll degradation”, Annual Review of Plant

Physiology and Plant Molecular Biology, Vol. 50 No. 1,

pp. 67-95.

Richardson, H. (1997), Handbook of Copper Compounds and

Applications, M. Dekker, New York, NY, pp. 93-121.

Somchit, M., Reezal, I., Nur, I. and Mutalib, A. (2003),

“In vitro antimicrobial activity of ethanol and water

extracts of Cassia Alata

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Starr, C., Evers, C. and Starr, L. (2011), Biology: Concepts and

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Usop, R., Abidin, Z., Mazni, N., Hadi, A., Halim, N., Taha, R.,

Careem, M., Majid, S. and Arof, A. (2016),

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Document Outline

Dual functions of Cu(NO3)2 as antifungal and colour stabilizer for coating paint film consisting of chlorophyll
- Introduction
- Experimental
  - Dye preparation
  - Preparation of coating paint film sample
  - Preparation of fungus sample
- Characterizations
  - Colour meter measurement
  - Gloss measurement of the coating sample
  - Zone of inhibition
- Results and discussion
  - Coating paint film sample preparation
  - Colour meter measurement
  - Gloss measurement
  - Zone of inhibition
- Conclusion
- References

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