Study of Free Radical Scavenging Activity on Film with Addition of

Silver Nanoparticle Synthesized using Papaya Leaves and Fruits

Eveline

, A. Herry Cahyana

, and Jessica

Lecturer, Food Technology Department, Faculty of Sains and Technology, University of Pelita Harapan,

Jl. MH. Thamrin Boulevard 1100 Lippo Village, Kelapa Dua, Karawaci, Tangerang, Indonesia

Senior Lecturers, Chemistry Department, Faculty of Mathematics and Natural Science, University of Indonesia,

Jl. Margonda Raya, Beji, Pondok Cina, Depok, Indonesia

Alumnus, Food Technology Department, Faculty of Sains and Technology, University of Pelita Harapan,

Jl. MH. Thamrin Boulevard 1100 Lippo Village, Kelapa Dua, Karawaci, Tangerang, Indonesia

Keywords: AgNPs, antioxidants, Carica_papaya, free_radical_scavenging, nanoparticles

Abstract: Silver nanoparticles (AgNPs) have been the focus of research in terms of catalyst, antimicrobial, and

biomaterial production due to the potential of proteins, amino acid residues, free radical anions (antioxidants),

and eukaryotic cell receptors. The synthesis of AgNPs using plant extracts such as leaf and papaya fruit is one

of the safest nanoparticles and has a wide metabolite for reduction, so it is timely to apply to food packaging

materials that have antioxidant activity. Initially, phytochemical compounds (phenolic and flavonoids) of

papaya leaves and fruit were each extracted with air and ethanol solvents (1:0, 1:1, 0:1). The papaya leaf was

selected as a better source of phytochemical compounds than papaya, while ethanol solvent was determined

as the best solvent, based on analysis on plant extract (solids content, total phenolic, total flavonoids,

antioxidant activity), and on AgNPs synthesis analysis (yield, level of inhibition, and characteristics of

AgNPs) with successive results of 1.52%, 659.65 mg GAE/g, 614.04 mg QE/g, 130.3 ppm, 15.76%, 82.44%,

and morphology according to nanoparticle criteria (round; 97.92 nm [SEM]; 79.92 nm [PSA]). AgNPs were

selected then homogenized with AgNPs nanoparticles (0.0, 0.4, 0.6, 0.8, 1.0%). The addition of 1%

nanoparticles gave the best characteristics of free radical resistant packaging films (4.5% starch and 1.5%

glycerol), 1.52 MPa tensile strength, 22.37% elongation, and 18.94% free radical inhibition (42 times greater

than control).

1 INTRODUCTION

Nanoparticles is a nanotechnology development that

has been interesting in recent years in various fields.

Not only in the fields of physics, chemistry, biology

and engineering, but also applications in the

environmental, biomedical, electronic, and optical

fields (Wahyudi, et al., 2011). Shefar (2007) added

that in the biomedical field, nanoparticles can

facilitate the entry of drug compounds into micro or

nanometer-sized tissue through blood transport,

manipulate temperatures when hypothermia, and

utilization in technique of Magnetic Resonance

Inmaging (MRI).

In addition to gold nanoparticles, silver

nanoparticles (AgNPs) are nanoparticles that are

often used in research. AgNPs have higher absorption

molar coefficients than other nanoparticles such as

iron, zinc, platinum, palladium nanoparticles. Optical

properties and measurements of AgNPs with better

UV-Vis spectrophotometers (Caro, et al., 2010). The

synthesis of AgNPs is increasingly developed

through chemical reduction that is environmentally

friendly, harmless, and relatively inexpensive, using

plant extract as a reducing agent (Sathishukumar, et

al., 2009).

Because of its potential as a micro particle that is

considered safe for the health of the body and

environmentally friendly, AgNPs are recommended

to also play a role in the field of food, such as the

manufacture of free radical food packaging products.

An antioxidant compound as an antidote to free

radicals inserted in nanoparticles package can be

extracted from leaves and papaya fruit (Carica

papaya L.). According to Duthie (2000), Zuhair, et al.

(2013), and Philip, et al., (2011); organic compounds

including phytochemical compounds that are

Eveline, ., Cahyana, A. and Lesmana, J.

Study of Free Radical Scavenging Activity on Film with Addition of Silver Nanoparticle Synthesized using Papaya Leaves and Fruits.

DOI: 10.5220/0010038600780083

In Proceedings of the 3rd International Conference of Computer, Environment, Agriculture, Social Science, Health Science, Engineering and Technology (ICEST 2018), pages 78-83

ISBN: 978-989-758-496-1

antioxidants in the plant part have the ability to be a

metal ion agent in the biosynthesis process. Jain, et al.

(2009) and Banala, et al. (2015) added that papaya

leaves can be a good source of free radical-free

compounds in the synthesis of AgNPs.

Handayani, et al., (2010) in his research extracted

antioxidant phytochemical compounds by using

water solvents (polar). Therefore, in this study, it is

designed that the extraction of plant phytochemicals

is used in different solvents of polarity to study if

other phytochemicals exist in plants that are insoluble

in water polarity and may be reducing agents in AgNP

synthesis; so the solvent used is water and ethanol

with a ratio of 1:0, 1:1, and 0:1. Analysis of plant

extract (solid content, total phenolic, total flavonoid,

antioxidant activity) and analysis of AgNPs (yield,

inhibition level, and AgNPs characteristics) were

performed to determine the best solvent ratio so that

further AgNPs of the selected ratio could be applied

in the manufacture of packaging film at the next

research stage.

AgNPs of the ratio yielding the best analysis were

then applied to the creation of the packaging film

(4.5% cassava starch and 1.5% glycerol) with

concentrations of 0.0, 0.4, 0.6, 0.8, 1.0%. The best

concentration determination was performed based on

tensile strength analysis, elongation, antioxidant

activity. The existence of this study is expected to be

a study that can continue to move the development of

nanoparticles not only in the area of food packaging

but also able to develop added value in other areas of

food technology.

2 MATERIALS AND METHODS

2.1 Materials

The materials: leaf and papaya fruit (Carica papaya

L.), methanol, distilled water, distilled water, tissue

paper, AgNO3 powder, glycerol, aluminum chlorite

2%, sodium hydydroxide solution (NaOH), and

Diphenyl-picryl-hydrazyl (DPPH).

2.2 Methods

The research consists of two stages (stages 1 and 2)

and is preceded by the sample preparation stage. The

sample preparation stage was performed to prepare

dried papaya leaves and fruit to be used in the

extraction, including: leaching each 30 grams of

leaves and papaya fruit with distilled water, cutting,

drying in the dryer cabinet (30°C, 24 hours), sieving

(40 mesh shifter).

Phase 1 study (Figure 1) began with the extraction

of dried papaya leaves and fruit (30 grams each) with

water solvent and ethanol ([1:0, 1:1, 0:1],

sample:solvent 1:8); extraction (Microwave Assited

Extraction [MAE], 450 watts, 13.5 minutes);

filtration (Whatman No. 1) resulting in extracts. The

analysis of the extract was carried out, ie: solids

content, total phenolic (Handayani, et al., 2011), total

flavonoids (Handayani, et al., 2014), antioxidant

activity (Nahak and Suhu, 2011 with Modification).

Meanwhile, 50 ml of AgNO

0.6 M solution was

prepared by dissolving 5 grams of AgNO

powder in

50 ml of distilled water. The extract and the AgNO3

solution were then homogenized (pH 8, 75-80°C, 3

hours) and centrifuged (5000 rpm, 15 min), the

resulting precipitate was applied to the watch glass

and dried (oven, 24 h, 60°C) AgNPs. AgNPs analysis

was performed: level of inhibition, and characteristics

of AgNPs (Scanning Electron Microscopy [SEM]

(Jain et al., 2009) and Particle Size Analyzer [PSA]).

Analysis of extracts and AgNPs is the basis for

determining the solvent ratio that produces AgNPs

with the best antioxidant activity with characteristics

appropriate to nanoparticle criteria according to

Albert et al. (2006).

In Phase 2 of the study (Figure 2), the synthesis of

AgNPs with the highest free radical retardant activity

in the first phase of the study was made as a silver

nanoparticles agent (0, 0.4, 0.6, 0.8, 1.0%) in the

making of free radical scavenging activity film (4.5%

cassava starch and 1.5% glycerol). Analysis of

inhibition ability (Nahak and Suhu, 2011 with

Modification) was performed to determine the best

concentration of AgNPs. Films with best inhibition

ability were then analyzed for tensile strength and

elongation (Zhong and Xia, 2008).

2.3 Experimental Design

The experimental design of phase 1 was a 1-factor

(solvent) Randomized Complete Design (solvent)

containing 3 levels (water, water: ethanol, ethanol 1:0

[A1], 1:1 [A2], 0:1 [A3]) with repetition 2 times. In

stage 1, a t-test of the two independent samples was

obtained which was the statistically highest yield on

each parameter of analysis to determine one part of

the plant (leaf or fruit) that had the best antioxidant

activity and characteristics as AgNPs. The Phase 2

study used Completely Randomized Design 1 factor

(AgNPs concentration) consisting of 5 levels (0.0

], 0.4 [A

], 0.6 [A

], 0.8 [A

], 1.0% [A

]) with 2

times of repetition.

Study of Free Radical Scavenging Activity on Film with Addition of Silver Nanoparticle Synthesized using Papaya Leaves and Fruits

Figure 1. Phase 1 Research Flowchart

Figure 2. Phase 2 Research Flowchart

3 RESULT

3.1 Phase 1

Phase 1 study was conducted to determine the

extraction solvent (water and ethanol 1:0, 1:1, 0:1)

and to determine the part of the plant (leaf or fruit)

that can produce the best free radical activity of

AgNPs. The analysis of the extract (solid content,

total phenolic, total flavonoid, antioxidant activity)

and AgNPs analysis (inhibitory level) were

performed to achieve these objectives, the AgNPs

characteristics of selected plant and solvent sections

were then analyzed SEM and PSA.

The result of statistical test showed the influence

of solvent type to the solid content both on leaves and

papaya fruit (p<0.05). Table 1 shows that the solids

of the extract with the ethanol solvent were the

lowest, both on the leaves and on the papaya fruit

(1.52 and 0.79%). The presence of water in a solution

can increase the yield due to components other than

extracted phenolics, protein solubility and

carbohydrates in water (Zielinski, 2016). Statistical

results with t-test showed that there were differences

of solids content in leaf and fruit extract (p<0.05).

According to Saran and Choudhary (2013), papaya

fruit contains thiamine, riboflavin, nicain, tryptophan,

methionine, lysine, magnesium, and phosphoric acid

not contained in papaya leaves as a contribution of

solids to the extract.

The results of statistical test also showed that the

solvent ratio had an effect on phenolic content, both

on leaf extract and papaya fruit (p<0.05). Table 1

shows that ethanol solvent yields the highest total

Synthesis of AgNPs with the highest free radical

activity in the first phase of the study

Silver nanoparticles agent (0, 0.4, 0.6, 0.8, 1.0%)

in the making of free radical scavenging activity

film

(4.5% cassava starch, 1.5% glycerol)

Analysis:

- Inhibition ability

Films with best inhibition ability

Analysis:

- Tensile strength

- Elon

ation

Dried papaya leaves and fruits

(30 grams each)

Extraction with water solvent

and ethanol ([1:0, 1:1, 0:1],

sample:solvent 1:8)

Extraction using MAE,

450 watts,

13.5 minutes)

Filtration using Whatman No. 1

AgNO

powder

5 grams

Dissolving with

50 ml distilled water

50 ml of AgNO

0.6 M solution

AgNPs

Analysis

- Solids content

- Total phenolic

- Total flavonoids

- Antioxidant activity

Centrifugation

(5000 rpm, 15 min)

Homogenization

(pH 8, 75-80°C, 3 hours)

Resulting precipitate was applied to the

watch glass and dried (oven, 24 h, 60°C)

extracts

Analysis

- Level of inhibition

- Characteristicsof AgNPs

- Particle Size Analyzer

ICEST 2018 - 3rd International Conference of Computer, Environment, Agriculture, Social Science, Health Science, Engineering and

Technology

phenolic in papaya leaf and papaya extracts compared

to water solvents and water-ethanol mixtures (659.65

and 245.12 mg GAE/g). The ethanol solvent is a

semi-polar solvent which is advantageous for

phenolic and flavonoid extracts (Chirinos, 2007). The

results of t-test on total phenolic leaf and papaya fruit

revealed that papaya leaf extract contained higher

total phenolic than fruit extract (p<0.05). According

to Johnson, et al. (2008) papaya leaves contain

alkaloid components, antokuinon, catechol,

flavonoid, phenolic, saponin, steriods, tannins,

triterpenoids, polyphenols, pholenicins are

compounds that contribute to the total phenolics of a

material.

The result of statistic test showed that the solvent

ratio influenced the total flavonoids of leaf extract

and papaya fruit (p<0.05). Table 1 shows that ethanol

solvent yields leaf extract and papaya fruit with the

highest total flavonoids (614.04 and 135.48 mg

QAE/g). Wang, et al. (2008) and Velioglu, et al.

(1998) revealed that papaya leaves and fruits contain

biologically soluble flavonoids in ethanol because

flavonoids correlate to total phenolics due to

flavonoid dominance of phenolic groups in papaya

leaf and papaya extracts. T-test between leaf extract

and papaya fruit showed papaya leaf extract

containing more flavonoids than papaya (p<0.05).

Most of the biological activity of papaya leaves is

contributed by kaemferol and quercetin which are

more soluble in ethanol (Maisarah, et al., 2013).

The result of statistic test showed that the solvent

ratio had an effect on the free radical activity of

papaya leaf extract and papaya fruit (p<0.05). Table

1 shows the ethanol solvent yielding the lowest IC

value of both the leaf extract and papaya fruit (130.3

and 508.65 ppm). Do, et al. (2014) in his study also

found that ethanol solvents gave the lowest IC

values compared to the use of other solvents. This

means, ethanol solvents are able to extract more

compounds that potentially counteract free radicals.

Lie, et al. (2006) and Hou, et al. (2003) stated that

phenolic and flavonoid components contain hydroxyl

acting as proton donors in free radical (antioxidant)

retention. T-test of antioxidant activity of leaf extract

and papaya fruit showed leaf extract has greater

potential to counteract free radical, IC

value is

smaller than IC

papaya fruit. This is related to the

total phenolic and flavonoid yields that are also

higher in papaya leaf extracts, both of these

compounds contribute to the antioxidant activity of a

substance. Hanani, et al. (2005) added that if a

material has an IC50 value <200 g/ml, then the

antioxidant activity is strong.

The result of statistical test showed that the yield

of AgNPs was influenced by the solvent ratio of both

the leaf extract and the papaya fruit (p<0.05). Table 1

shows the leaf extract from the ethanol solvent

yielding the lowest yield of AgNPs (15.76%), while

the yield of AgNPs reached the highest value on fruit

extract (7.39%). T-test between yield of extract on

leaf and fruit showed that papaya leaf yield higher

rendement than papaya fruit (p<0.05). Phenolic

content and flavonoid in leaves are higher than in

fruits thus increasing the strength of Ag

reduction in

the process of forming nanoparticles and increasing

the number of nanoparticles formed (Do, et al., 2014).

Table 1. Phase 1 Test Result

Parameters

Wate

:Ethanol

Leaves Extract Fruit Extract

1:0 1:1 0:1 1:0 1:1 0:1

Solid Content

(

)

3.61±0.95

3.80±0.38

1.52±0.15

4.53±0.14

4.64±0.21

0.79±0.18

Total Phenolic (mg

GAE/g)

86.86±2.05

137.21±4.49

659.65±5.62

15.81±1.09

19.22±0.50

245.12±3.18

Total Flavonoid (mg

QAE/

97.79±0.98

153.18±2.00

614.04±4.07

57.21±2.10

108.29±1.27

135.48±2.86

(ppm) 278.42±6.00

210.63±6.65

130.30±0.90

1139.21±7.04

912.85±7.33

508.65±8.57

Yiel

(%) 10.66±0.39

13.11±0.41

15.76±0.45

3.39±0.37

5.67±0.39

Inhibition

(

)

40.95±1.21

55.88±0.95

82.44±0.66

36.52±1.37

43.66±1.33

53.17±0.22

Note: - Different notation showed there was significant difference (p<0.05)

- No comparison between parameter analysis

- Different notation not showed t-test result

The result of statistic test of solvent type shows

the solvent ratio influence the inhibition level of leaf

extract and papaya fruit (p<0.05). Table 1 shows the

two extracts with ethanol solvent having the highest

rates of inhibition compared to other solvents, both in

leaf extract and fruit extract (82.44 and 53.17%). This

is associated with greater antioxidant activity

resulting in extraction with alcohol solvents than

other solvents. According to Ahmad and Sharma

(2012), the mechanism of free radical retardation

Study of Free Radical Scavenging Activity on Film with Addition of Silver Nanoparticle Synthesized using Papaya Leaves and Fruits

activity starts from AgNPs cation which gets

electrostatic appeal from bioactive components of

plant extracts as a result of reduced cations, while the

phytochemicals covered by AgNPs and their

bioactivity rose synergistically:

AgNO

→ Ag

+ NO

; e + Ag

→ Ag

T-test inhibition level of leaf extract and papaya fruit

showed papaya leaf extract had greater inhibition rate

than fruit (p<0.05). It is associated with leaf extracts

that have a potent antioxidant activity greater than

fruit.

Based on the analysis of AgNPs extract and

nanoparticles, it was determined that nanoparticles

with papaya leaf extract extracted by ethanol have the

potential to counteract free radicals better than other

solvents. AgNPs were then analyzed using SEM and

PSA characteristics. The results of AgNPs

characteristic test with SEM showed agrous

morphology of 97.92 nm, spherical, and there were

coagulated particles (Figure 3). Test with PSA is a

strengthening test of AgNPs characteristics with

SEM. In the PSA test, AgNPs appears to be 79.92 nm

in size (Figure 3). In both the SEM and PSA tests, the

size of AgNPs nanoparticles is not out of nanoparticle

size requirements according to Albert, et al. (2006),

which is 1-100 nm.

Figure 3. AgNPs Morphology

3.2 Phase 2

The second phase of the research is the application

stage of AgNPs that has been synthesized in the

previous stage on the free radical container

packaging. The packaging film material formulation

comprises 4.5% starch, 1.5% glycerol, and the best

AgNPs in the addition of 0.0, 0.4, 0.6, 0.8, 1.0%. The

statistical test showed the effect of AgNPs

concentration on free radical inhibition of packaging

film (p<0.05). Table 2 shows a concentration of

AgNPs of 1% capable of inhibiting the largest free

radical (18.94%) and increased by 42 times compared

to control (0.45%). The higher concentration of

AgNPs is added, the film structure is more compact

and flexible (not rigid), and clear.

AgNPs with 1% concentration were then analyzed

tensile strength and elongation with consecutive

values were 1.52±0.07 Mpa and 22.37±4.12%.

Warkoyo (2014) said a film may have a strong

breakup if it is 31.65%. Therefore, further research is

needed on better formulations, especially of the type

of material and the concentration of hydrocolloids,

lipids, and edible composite films of free radical in

order to achieve the value.

Table 2. Phase 2 test Result

Concentration of A

NPs (%) Inhibition (%)

0.0 0.45 ± 0.36

0.4 4.88 ± 0.77

0.6 8.17 ± 0.63

0.8 14.77 ± 0.35

1.0 18.94 ± 0.36

Note: - Different notation showed there was

significant difference (p<0.05)

4 CONCLUSIONS

The papaya leaf with ethanol solvent was chosen as a

source of phytochemical and solvent compounds

capable of producing the best free radical extract and

AgNPs based on 1.52% solids, total phenolic 659.65

mg GAE/g, total flavonoids 614.04 mg QE/g,

antioxidant activity 130.3 ppm, AgNPs 15.76%, level

of inhibition AgNPs 82.44%, and morphology

according to nanoparticle criteria (round, 97.92 nm

[SEM]; 79.92 nm [PSA]). As much as 1% of the best

AgNPs are able to produce free radical-free films

with 1.52 MPa tensile strength, 22.37% elongation,

and able to inhibit free radicals by 18.94% (up 42

times compared to controls).

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Study of Free Radical Scavenging Activity on Film with Addition of Silver Nanoparticle Synthesized using Papaya Leaves and Fruits