Characterization and Antibacterial Activity of Dayak Onion

(Eleutherine palmifolia) Hydrogel in Vitro

Uswatun Chasanah

, A. B. Apriliyanto, D. Anggara, Ayu Kusumawardani, and Dian Ermawati

Department of Pharmacy, Faculty of Health Science, University of Muhammadiyah Malang, Jalan Bendungan Sutami 188A

Malang 65145, East Java, Indonesia

Keywords: Eleutherine palmifolia, Hydrogel, Antibacterial, Staphylococcus aureus, Staphylococcus epidermis,

Pseudomonas aeruginosa.

Abstract: Traditionally, the crushed bulbs of Dayak onion (Eleutherine palmifolia) used for wound healing due to

naphthoquinone compounds found in the bulbs of Dayak onion can inhibit the growth of Staphylococcus

aureus, Staphylococcus epidermis, and Pseudomonas aeruginosa that cause skin infection. To improve the

ease of using the bulbs of Dayak onion as drugs for the treatment of wounds that need to be made in the

appropriate dosage form is a topical hydrogel, hydrogels contained Dayak onion extract at a level of 2%,

4.5%, and 6.0% were made then determined its characterization which included organoleptic, pH, viscosity,

and spreadability, determined of antibacterial activity by a well-diffusion method. The higher levels Dayak

onion extract resulted in hydrogels which homogenous, sharp color intensity, more aromatic, decreased

viscosity, and increased spreadability while for pH was not different, while the highest antibacterial activity

achieved by Dayak onion hydrogels 6.0% which had antibacterial effectiveness to the Staphylococcus aureus,

Staphylococcus epidermis, and Pseudomonas aeruginosa were 59.2%, 49.5%, and 63.1%, respectively, and

as control was tetracycline HCL 1.0%. Thus, the Dayak onion hydrogels 6.0% is the best.

1 INTRODUCTION

Dayak Onion (Eleuherine palmifolia) is a plant

belonging to the Iridaceae family which originated

from tropical America and has been widely cultivated

in Central Kalimantan (Indonesia), South China,

Thailand, and South Africa(Insanu, Kusmardiyani, &

Hartati, 2014). Empirically, Dayak onion has been

used by local people as a cure for various types of

diseases such as breast cancer, hypertension, diabetes

mellitus, cholesterol, ulcer, colon cancer and

preventive stroke (Galingging, 2009). In another

place, these plants are used for wound healing

(Agyare et al., 2009).

The phytochemical screening showed the bulb

extract of Dayak onion contained flavonoids,

polyphenols, alkaloids, quinones, tannins, steroids,

monoterpenoids, and sesquiterpenoid (Puspadewi,

Adirestuti, & Menawati, 2013) which the presence of

antibacterial activity on onions is due to the

naphthoquinone(Kuntorini & Nugroho, 2010). It was

reported the ethanol extract of Dayak onion can

inhibit the growth of gram-negative and gram-

positive bacteria strain included Staphylococcus

epidermis (Novaryatiin, Pratiwi, & Ardhany, 2018),

Staphylococcus aureus, and Pseudomonas aeruginosa

(Padhi & Panda, 2015) that are normal microbiota on

the skin (Byrd, Belkaid, & Segre, 2018) and have

opportunistic property, so, they easily enter the body

if there is an open wound and cause skin

infections(Cohen et al., 2016) (Niazi et al., 2010)

(Wong, Rodrigue, & Kassen, 2012).

Traditionally, Dayak onion has been used for

wound healing by applying crushed bulbs of Dayak

onion to the wound (Agyare et al., 2009). This is of

course not practical, and therefore needs to be

developed into an appropriate topical preparation in

the form of hydrogel preparations.

The hydrogel is semisolid systems consisting of

dispersions of small or large molecules in water

rendered jelly-like by the addition of a gelling

agent(Allen, Popovich, & Ansel, 2012). Some of the

main advantages of hydrogel formulations over other

semisolid dosage forms are the hydrogel easily

formulated, elegant, non-greasy, easily applied,

biodegradable and biocompatible, forms a protective

coating, provides a cool sensation, and it is suitable

for both polar and non-polar drugs(Kaur & Guleri,

164

Chasanah, U., Apriliyanto, A., Anggara, D., Kusumawardani, A. and Ermawati, D.

Characterization and Antibacterial Activity of Dayak Onion (Eleutherine palmifolia) Hydrogel in Vitro.

DOI: 10.5220/0009126201640170

In Proceedings of the 2nd Health Science International Conference (HSIC 2019), pages 164-170

ISBN: 978-989-758-462-6

2013), moreover, hydrogels have a relatively high

water absorption that is very useful for absorbing

exudates from wet sores, therefore using of hydrogel

base for topical preparations, especially for dealing

with acne, atopic dermatitis or wounds is very

appropriate.

Purpose of this research was to made hydrogels

containing Dayak onion extract at various

concentrations, then the determined of

characterization which includes organoleptic, pH,

viscosity, homogeneity, and spreadability, continued

the antibacterial assay against Staphylococcus aureus,

Staphylococcus epidermis, and Pseudomonas

aeruginosa by the wells-diffusion method.

2 METHODS

Materials.

Active Ingredients: Dayak onion bulbs obtained from

Tabalong, South Kalimantan, Indonesia which had

been determined by the Materia Medica Technical

Services Unit, Batu, East Java Province Health

Office, Indonesia.

Excipient Ingredients: Carbopol (Fagron Hallas,

Greece), glycerin, propylene glycol (Dow Chemical

Co.), triethanolamine, methylparaben (Ueno Fine

Chemical, Japan); pure water, Oxytetracycline HCl.,

Mueller Hinton Agar (MHA) Media, Na Cl 0.9%.

Research Procedure.

Prior making of ethanol extract of Dayak onions uses

the maceration method. The formula of hydrogel

contained active ingredients extracts of Dayak onions

at a concentration of 2%, 4%, and 6 %. The

determined of physical-chemical characteristics

carried out which includes organoleptic,

homogeneity, pH, viscosity, and spreadability, while

the antibacterial activity of the Dayak onion hydrogel

tested against Staphylococcus aureus,

Staphylococcus epidermis, and Pseudomonas

aeruginosa using agar well diffusion method and as a

positive control, is Oxytetracycline HCl.

Preparation of Ethanol Extract of Dayak Onion.

A Dayak onion extract made by maceration using

96% ethanol. A total of 1.5 kg dry of Dayak onions

made powder, the powder soaked in 5.0 liters of

ethanol 96% for 24 hours, it separated between the

filtrate and the pulp then the filtrate that contains

ethanol, it evaporated with a vacuum rotary

evaporator at a temperature of 55°C to obtain a thick

extract of Dayak onions.

Preparation of Dayak Onion Hydrogel.

Prepared carbopol hydrated, added methylparaben

which has been dissolved in propylene glycol then

glycerin. This mixture dropped with triethanolamine

until a neutral pH. Furthermore, added Dayak onion

extract which has been dissolved in propylene glycol

and then remaining water. The composition of the

Dayak onion hydrogel formulation as shown in Table

Determined Ethanolic Extract of Dayak Onion.

Determined ethanolic extract of Dayak onion

included the characteristic (consistency, smell, and

color) and pH.

Determined of physical-chemical characteristic

Evaluation of the Dayak onion hydrogel is a physical

and chemical characteristic. Determined physical

characteristics include organoleptic, homogeneity,

viscosity, and spreadability, whereas chemical

characteristic test is pH.

Physical Characteristic.

Organoleptic. Organoleptic determination of gel

products carried out by visually including color, odor,

and consistency.

Homogeneity. Weighed of 0.1 gram of hydrogel

spread evenly and thinly on the object-glass. The gel

must show a homogeneous arrangement and there are

no spots.

Determined of Viscosity.

Measurement of gel viscosity carried out using a

Brookfield Viscometer. A total of 150 grams of the

hydrogel remained into the beaker glass. The spindle

no.4 installed, then it lowered until immersed into a

gel product. The speed on the device is the start from

0.3 rpm to 60 rpm. On each measurement with a

difference in speed, the scale reads until the moving

red needle has stabilized.

Spreadability.

The spread test is performed to determine the ease of

preparation when applied to the skin. Weighed 0.5 g

hydrogel which is placed in the middle of a large

round glass. On top of the hydrogel is placed a

transparent round glass that has been weighed

beforehand and left for 1 minute. The dispersing

hydrogel is measured in diameter by taking the

average length of the diameter from several sides.

Characterization and Antibacterial Activity of Dayak Onion (Eleutherine palmifolia) Hydrogel in Vitro

165

Table 1: The Composition of Dayak onion hydrogel.

Materials

Composition (%)

Dayak Onion Extract

Carbopol

Tri ethanolamine

Propylene glycol

Glycerin

Methylparaben

0.1

Pure water to

100

*) is sufficient to neutral.

Above it given a load of 50 grams, left for 1

minute and recorded the distribution diameter.

Continue to increase the load each time by 50 grams,

after 1 minute the distribution diameter recorded until

a sufficient diameter obtained to see the effect of the

load on the change in the diameter of the gel spread.

Chemical Characteristic: pH.

The determination of pH is done using a pH meter.

Previous calibrating the device with a buffer solution

of pH 4.0 and pH 7.0. The pH determination carried

out by dipping the pH meter electrode into the

hydrogel, observing the number printed on the

monitor. A good hydrogel must have a pH that

matches the skin's pH which is 4-6.5(Ali &

Yosipovitch, 2013).

Determined of Antibacterial Activity.

Antibacterial activity of each formula of Dayak onion

hydrogel carries out by the agar well diffusion

method. The pathogenic bacteria used were

Staphylococcus aureus and Staphylococcus

epidermis obtained from the Laboratory of

Biomedical, University of Muhammadiyah Malang,

Indonesia, and Pseudomonas aeruginosa obtained

from Laboratory of Microbiology, Brawijaya

University, Malang, Indonesia. Each antibacterial

assay was carried out in three replicated. As a

negative control was a gel base and as a positive

control was Oxytetracycline ointment 1.0%.

All test bacteria cultured in Mueller Hinton Agar

(MHA) Media for overnight at 37 C before used for

the antibacterial assay. Antibacterial assay was

carried out as follows. A 6 mm diameter well was

made on the Mueller Hinton Agar medium which had

been inoculated of the bacterium. Dayak onion

hydrogel 2.0%, 4.0%, and 6.0%, as well as positive

control and negative control, added as much as

0.10gram in the hole. These incubated for 1x24 hours

at 37ºC then the diameter of inhibition zone observed

after the incubation period. The inhibitory activity

calculated by equation (1):



















 (1)

Where d1 is the diameter of well (6mm) and d2 is the

diameter of the inhibition zone(mm).

Then, the result of the measurement of inhibition

zone of each gel formula was calculated its

antibacterial effectiveness by comparing the diameter

of inhibition zone each gel formula with that of

positive control, oxytetracycline ointment 1.0%.

The antibacterial effectiveness of hydrogel

formulation to the positive control was calculated by

equation (2) (Oroh, Kandou, Pelealu, & Pandiangan,

2015):

 





 (2)

Where D is a diameter of inhibition zone of hydrogel

(mm) and Da is a diameter of inhibition zone of

positive control (mm).

Data Analysis.

The organoleptic examination is done one day after

making by visual means to observe the preparation

directly including texture, odor, color, and taste.

Meanwhile, physical-chemical characteristics

including pH, viscosity, spreadability as well as

inhibitory activity test result were analyzed using a

one-way ANOVA test at a degree of confidence α =

0.05 using the SPSS 16.0 program. If the results of P

<α, the analysis is continued by the Least Significant

Difference (LSD) or Honestly Significant Difference

(HSD) test to find out where the different data are.

3 RESULTS AND DISCUSSION

Characteristic of Ethanol Extract of Dayak

Onions.

Ethanol extract of Dayak onions obtained from the

extraction process by maceration method using 96%

ethanol is a thick liquid that has a strong brownish-

red and aromatic, and the result of pH determined

hows it has a pH of 5. So it is acidic property.

HSIC 2019 - The Health Science International Conference

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Table 2: Physical-chemical characteristics of Dayak onion hydrogels.

Formula

Organoleptic

Homogeneity

Viscosity (P˖as)

Spreadability

(g/cm)

speed

0.3rpm

0.6rpm

1.5rpm

Semisolid, soft,

aromatic, brownish-

red color

homogen

6.31±0.10

753±110

460±65

300±24

0.0040±0.0000

Semisolid, soft,

aromatic, brownish-

red color

homogen

6.12±0.21

633±23

437±15

281±38

0.0043±0.0001

Semisolid, soft,

aromatic, brownish-

red color

homogen

5.97±0.28

520±2

386±3

217±8

0.0050±0.0000

Physical-chemical Characteristics of Dayak Onion

Hydrogels.

All are hydrogels that have a flat texture, the same

color and no coarse particles in it. They had a soft,

aromatic, and brownish-red color whose color

intensity according to the levels of extract in each

formula, the higher the extracted content, the sharper

the color (Figure 1).

The physical-chemical characteristic of hydrogels

as shown in Table 2. The pH of hydrogels containing

Dayak onion 2.0%, 4.0%, and 6.0% each have a pH

of 6.31, 6.12, and 5.97.

Viscosity determination by a Brookfield

Viscometer uses a spindle number of 64 at speeds of

0.3 rpm., 0.6 rpm., and 1.5 rpm. The results of

determining the viscosity of hydrogel 2.0%, 4.0%,

and 6.0% at each speed as shown in Table 2. The

viscosity of hydrogel 2.0%, 4.0%, and 6.0% at 0.3 is

753P˖as, 633P˖as, and 520P˖as, respectively; at

0.6rprpm is 460P˖as, 437P˖as, and 386P˖as,

respectively; at 1.5rpm is 300P˖as, 281P˖as,

respectively, while the spreadability each has of

0.0040g/cm, 0.0043g/cm, and 0.0050g/cm.

Figure 1: Hydrogel of Dayak Onion at a Concentration of

2.0% (F1), 4.0% (F3), and 6.0% (F3).

Antibacterial Activity.

Anti-bacterial test results showed that the hydrogel of

Dayak onion can inhibit the growth of

Staphylococcus aureus, Staphylococcus epidermis,

and Pseudomonas aeruginosa as shown in Figure 2, it

appears that the largest inhibition zone occurs in

Staphylococcus epidermis while to the

Staphylococcus aureus and Pseudomonas aeruginosa

are not different.

The result calculation of inhibitory activity and

antibacterial effectiveness can be seen in Table 3.

The inhibitory activity to the growth of

Staphylococcus aureus of hydrogel 2.0%, 4.0%, and

6.0% is 0.0%, 63.3%, and 96.7%; to the growth of

Staphylococcus epidermis is 140.7%, 144.4%, and

196.3%; and to the growth of Pseudomonas

aeruginosa is 0.0%, 66.7%, and 107.4%. While using

oxytetracycline 1.0% as a standart the result of

antibacterial effectiveness hydrogel 2.0%, 4.0%, and

6.0% to the Staphylococcus aureus is 0.0%, 38.6%,

and 59.2%; to the Staphylococcus epidermis is

35.5%, 36.5%, and 49.5; to the Pseudomas

aeruginosa is 0.0%, 39.3%, and 63.1%.

(A)

(B)

(C)

Figure 2: Inhibition Zone Antibacterial Activity of Dayak

Onion Hydrogel 2.0% (F1), 4.0% (F2), and 6.0% (F3)

against S. aureus (A), S. epidermis (B), P. aeruginosa (C).

Characterization and Antibacterial Activity of Dayak Onion (Eleutherine palmifolia) Hydrogel in Vitro

167

Table 3: Antibacterial activity of Dayak onion hydrogel.

Bacteria

Inhibitory Activity %

S. aureus

0.0

63.3

96.7

S. epidermis

140.7

144.4

196.3

P.aeruginosa

0.0

66.7

107.4

Antibacterial effectiveness(%)*

S. aureus

0.0

38.6

59.2

S. epidermis

35.5

36.5

49.5

P. aeruginosa

0.0

39.3

63.1

*oxytetracycline 1.0% as a positive control

Physical-chemical Characteristics of Dayak Onion

Hydrogels.

Hydrogel preparations that made containing the bulb

of Dayak onion extracts at a concentration of 2.0%,

4.0%, and 6.0%. It found that all hydrogels are

homogenous, not greasy, aromatic red-brown that is

sharpener according to the level of the bulbs of the

Dayak used (Figure1). If the terms of organoleptic,

then all is good hydrogel formula.

The degree of acidity or pH of topical preparations

must be safe for the skin in the range of pH 4.1-5.8

(Proksch, 2018), so it does not cause skin irritation.

The results of the determined pH of Dayak onion

extract gel as shown in Table 2. The pH of the

hydrogel is more than 5.8, but this does not harm the

skin because the skin has a tolerance for exposure to

materials that have a pH of up to pH 8 (Kim et al.,

2009). The result of analysis using ANOVA One-way

at =0.05 was obtained p (0,270) > 0.05, it is means

all of these formulas have no different pH and are safe

for the skin. Thus the different levels of extract used

do not affect the pH of the gel produced even though

the extract is acidic (pH 5).

That the viscosity of the Dayak onion hydrogel

2.0% and 4.0% are not significantly different, but

these have a higher viscosity than Dayak onion

hydrogel 6.0%. This difference in viscosity may be

due to the acidic nature of the Dayak onion extract

that has a pH of 5. The gelling agent used in this

formulation is carbopol. The viscosity of carbopol

depends on pH, carbopol will have a good viscosity

at a neutral pH and will decrease at acidic pH

(Gibson, 2016).

Thus the higher the extracted content, the more

acidic the pH of the hydrogel preparation so that the

viscosity decreases. The curve profile in Figure 3

shows the viscosity of Dayak onion hydrogels is

different at each speedy, the higher the stirring speed,

or shear rate the lower the viscosity, which means the

flowing of Dayak onion hydrogel is pseudo-plastic

behavior (Kaur & Guleri, 2013).

Figure 3: The Viscosity of Dayak onion hydrogel at speed

variation.

The ease of spread of Dayak onion hydrogels,

when applied to the skin surface, is called

spreadability. The ability to spread is an important

characteristic in topical preparations because it

influences the transfer of active ingredients to the

target area in the right dosage and ease of use. It has

seen the spreadability of Dayak onion hydrogel 2.0%

and 4.0% were not significantly different,

respectively 0.040g/cm and 0.043g/cm, and both of

these were lower than Dayak onion hydrogel 6.0%

which had a spreadability of 0.05 g/cm. This result is

according to the viscosity of each gel formula that an

increase in viscosity will reduce the spreadability. So,

the Dayak onion hydrogel 6.0% is the best.

Antibacterial Activity.

Determination of antibacterial activity of Dayak

onion hydrogel against Staphylococcus aureus,

Staphylococcus epidermis, and Pseudomonas

aeruginosa by the agar well diffusion method,

antibacterial activity indicated by inhibition zone

formed around the well and the results of the

antibacterial activity test indicated that Dayak onion

hydrogel positively inhibit the growth of

Staphylococcus aureus, Staphylococcus epidermis,

and Pseudomonas aeruginosa as shown in Figure 2,

Dayak onion hydrogel 2.0% only have

antibacterial activity toward Staphylococcus

epidermis, it’s inhibitory activity is 140.7%, while

inhibitory activity Dayak onion hydrogel 4.0%

toward Staphylococcus aureus, Staphylococcus

epidermis, and Pseudomonas aeruginosa each is

63.3%, 144.4%, and 66.7%. And the inhibitory

activity of Dayak onion hydrogel 6.0% is 96.7%,

196.3%, and 107.4%, respectively. That increasing

the concentration of the extract correlated with an

increase in inhibitory activity except for

Staphylococcus epidermis where Dayak onion

hydrogel 2.0% and 4.0% have no different

antibacterial activity. Thus, the highest inhibitory

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activity of Dayak onion hydrogel is toward the

Staphylococcus epidermis.

Antibacterial effectiveness was calculated using

oxytetracycline ointment 1.0% as a positive control.

The results showed the effectiveness of antibacterial

Dayak onion hydrogel 2.0%, 4.0%, and 6.0% to the

growth of Staphylococcus aureus, Staphylococcus

epidermis, and Pseudomonas aeruginosa was lower

than the positive control, the effectiveness of

antibacterial Dayak onion hydrogel 6.0% to the

Staphylococcus aureus, Staphylococcus epidermis,

and Pseudomonas aeruginosa were 59.2%, 49.5%,

and 63.1%, respectively, Dayak onion hydrogel 4.0%

even lower, each is 38.6%, 36.5%, and 39.3% while

Dayak onion hydrogel 2.0% just to Staphylococcus

epidermis which its antibacterial activity is 35.5%.

The Dayak onion hydrogels 6.0% have antibacterial

effectiveness more than about 50%, so they were

categorized effectively.

4 CONCLUSIONS

Physical-chemical characteristics of Dayak onion

hydrogels at all concentrations are good while Dayak

onion hydrogel 6.0% is effective as an antibacterial

against Staphylococcus aureus, Staphylococcus

epidermis, and Pseudomonas aeruginosa.

ACKNOWLEDGEMENTS

The authors ' thanks to Joko Trisilo Wahono for his

dedicated work in collecting data used in this article

as a part of the objective of our research for

formulated and determined the activity of Dayak

onion hydrogel.

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