An Experiment for Extracted Citrus Hystrix Leaf Effectiveness on

Pityrosporum Ovale Fungi Growth

Sri Lestari Ramadhani Nasution

, Ali Napiah Nasution

and Sri Wahyuni Nasution

Department of Public Health, Faculty of Medicine, University Prima Indonesia, Medan

Department of Tropical Medicine, Faculty of Medicine, University Prima Indonesia, Medan

sriwahyuninasution@unprimdn.ac.id

Keywords: Effectiveness, Citrus Hystrix, Dandruff, Pityrosporum Ovale Mushroom.

Abstract: The growth of Pityrosporum ovale causes an inflammatory reaction both due to metabolite products that enter

the epidermis and fungal cells itself through the activity of T lymphocytes and Langerhans cells. This causes

discomfort and insecurity. Treatment of dandruff usually uses shampoos but some people often experience

allergic contact dermatitis due to the chemical side effects of these shampoos. Jeruk purut (Citrus Hystrix)

leaves are easily found in Indonesia and are natural ingredients that contain anti-fungal compounds, namely

flavonoids and tannins. This type of research was a laboratory experiment with the sensitivity test method of

the diffusion disk method. The sample used was 5 kg of citrus hystrix leaves and incubated for 48-72 hours.

This study used a concentration of 25%, 50%, 75%, the negative control contained aquadest, while the positive

control used 2% ketoconazloe. Experiments were carried out three times. The results showed that the highest

inhibition zone diameter was at a concentration of 75%, namely 16.20 mm.

1 INTRODUCTION

Jeruk purut (citrus hystrix) is a plant that is known to

the public as a food source and is thought to contain

active compounds which are believed to be herbal

medicine with very high antioxidant activity so that it

is widely used in daily needs, both in medical,

industrial, and household. The use of kaffir lime fruit

and leaves has been known by the public for a long

time as herbal medicine. Leaf and fruit parts are

usually used to overcome fatigue and improve body

fitness and as a flavoring for food (Adrianto, H.,

Yotopranoto, S. and Hamidah, H. 2014).

Citrus hystrix leaves are strongly suspected to

contain triterpenoid steroids, tannins (1.8%), and

essential oils (1-1.5%) so that they can be used as an

anti-fungal. The scalp contains saponins, tannins

(1%), and essential oils that contain citrate (2-2.5%)

(Santoso, 2015).

The fungus that causes dandruff is Malassezia sp.

and one of its species is Pityrosporum ovale. This

fungus is a normal flora that is in the hair, but various

conditions such as temperature, humidity, high oil

content, and decreased immunity can trigger the

growth of this fungus. (Alawiyah, Khotimah, and

Mulyadi, 2016).

Dandruff is a scalp problem that makes sufferers

feel disturbed both physically and psychologically. In

people with dandruff, the number of Pityrosporum

ovale will increase by more than 47%. Dandruff

occurs due to malfunctioning caused by changes in

the keratinization process which is constantly pushed

to the surface of the skin and becomes layered, dry,

brittle, and easily loose scales. (Karta and

Burhannuddin, 2017)

Malassezia sp is reported to be resistant to the use

of azole drugs. One way to cope with and prevent

excessive dandruff is to use anti-dandruff shampoo.

The levels of active substances in anti-dandruff

shampoos may also cause allergic skin reactions.

Active substances such as sulfur compounds,

selenium sulfide which are accumulated and absorbed

by hair follicles can also cause hair loss (Idris, 2013).

Citrus Hystrix leaf extract contains alkaloids,

flavonoids, terpenoids, and phenols which have

antioxidant activity. The most potent antioxidant

effects of Citrus Hystrix leaf extract are flavonoids,

alkaloids and phenols (Lawrence BM, 2018).

Based on the results of previous research, it was

found that the test results of the IC50 antioxidant

value of Citrus Hystrix leaf extract were 25.907 ±

0.187; ethosome of Citrus Hystrix leaf extract

Ramadhani Nasution, S., Nasution, A. and Nasution, S.

An Experiment for Extracted Citrus Hystrix Leaf Effectiveness on Pityrosporum Ovale Fungi Growth.

DOI: 10.5220/0010352902910295

In Proceedings of the International Conference on Health Informatics, Medical, Biological Engineering, and Pharmaceutical (HIMBEP 2020), pages 291-295

ISBN: 978-989-758-500-5

291

formula 1 of 28.814 ± 0.431; formula 2 is 32.299 ±

1.893 and formula 3 is 30.234 ± 0.531 which shows

that the three formulas have very strong antioxidant

activity (Kasuan N,et all , 2009).

2 METHOD

To determine the inhibition of citrus hystrix leaf

extract against the fungus Pityrosporum ovale, the

sensitivity test method (disc diffusion) was used. In

the experiment, 5 treatment groups included negative

control using aquadest, positive control using

ketoconazole ointment 2%, concentrations of 25%,

50%, and 75%. The medium used was Potato

dextrose agar. The research was conducted at the

Microbiology Laboratory of the Faculty of Medicine,

Prima Indonesia University.

The tools and materials used in the experiment

were petri dishes (7 pieces), test tubes, filter paper,

measuring cups, autoclaves, incubators, disc paper,

cotton swabs, tweezers, sterile cotton, calipers, drop

pipettes, erlenmeyer flasks, analytical scales. , test

tube racks, 5 kg kaffir lime leaves, 70% ethanol, 96%

ethanol, Pityrosporum ovale mushrooms, 2%

ketoconazole ointment, alcohol, aquabidest,

physiological NaCl, spiritus, potato dextrose agar

(PDA) media, plastic wrap, and aluminum foil.

Preparation stages, namely: The tools and

materials used are sterilized in an oven at a

temperature of 40-70º C for ± 2 hours, At this stage

of making citrus hystrix leaf water, first clean the

citrus hystrix leaves with water until clean then the

citrus hystrix leaf water is sliced small and blended

(Figures 1 and 2.

The citrus hystrix leaf extract which has been

evaporated with ethanol was divided into 5 treatment

groups with a concentration of 25%, 50%, 75%,

positive control, and negative control by dissolving

with 96% ethanol technique. Dissolving with ethanol

aims to remove the essential oil content in the extract.

The extract was made by macerating the citrus hystrix

leaves which had been cleaned with running water

before drying. The dried citrus hystrix leaves were

mashed using a blender to form simplicia powder.

From each simplicia, 1 kg of sample was taken and

then dissolved or soaked using 10 liters of absolute

ethanol (96% ethanol) solvent per each simplicia

(Figure 1). Then stirring per day for 1 hour. This

maceration process was carried out 3 times.

The citrus hystrix leaves are washed with running

water then drained until dry and put in a drying

cabinet for 3 days before mashed using a blender. The

powder was sifted to a fine powder of the citrus

hystrix leaves.

For the experiment, several tools such as petri

dishes (7 pieces), test tubes, measuring cups, are

sterilized first using an autocalved at a temperature of

121ºC for about 15 minutes. Meanwhile, the tools

made of metal are sterilized on an incandescent fire

for about 1 minute.

Preparation of Potato Dextrose Agar (PDA) as

much as 65 grams of medium is suspended in 1 liter

of distilled water into an erlenmayer flask and then

stirred using a hotplate strirer for 1 minute or until

dissolved. The sterilization was then carried out by

autoclave for 15 minutes at 121 ° C and allowed to

cool until the temperature has decreased to 40-45 ° C.

The results obtained were in the form of jelly poured

into a petri cloud and cooled until frozen.

The preparation of the Pityrosporum Ovale

Mushroom suspension used in this study was

obtained from the USU Pharmacy Laboratory. The

Pityrosporum Ovale used was made by taking a loop

of germs from the culture so that it is tilted and then

put into a tube containing physiological NaCl then

stirring until all fungal colonies dissolve in NaCl.

The diffusion test of pityrosporum ovale was

carried out by: PDA media that had hardened, evenly

scratched the entire surface of the media using a

cotton swab containing the suspension of

Pityrosporum Ovale fungus; a sterile blank disk is

taken; dipped in 50% concentration, and finally

placed on the surface of the media that has been

scratched by the fungus Pityrosporum Ovale. The

treatment is carried out for all concentrations then

spaced apart to prevent the inhibition zones from

forming. The same experiment was repeated 3 times

for data collection needs. All test isolates were

incubated for 36-48 hours at 37ºC in an incubator.

After 36-48 hours, the zone of inhibition is measured

using a caliper. The classification of the response to

fungal growth inhibition is given in Table 1

(Maryanti, Marta, Della, and Hamidy, 2017).

Table 1 Classification of fungal growth inhibition

responses.

Clear zone diameter

Inhibition Growth

Rresponse

>2cm

Very strong

1,6-2 cm

Strong

1-1,5 cm

Moderate

<1 cm

Weak

From Table 1, it is obtained that the larger the

diameter of the clear inhibition zone was > 2 cm, then

the response to fungal growth will be stronger and on

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the contrary, the response to growth resistance will be

weaker. In summary, the experimental process can be

described as in Figure 3.

Figure 1. Drying process of Citrus Hystrix and mashed

leaves

Figure 2. The process of filtering and making Citrus Hystrix

leaf water

Figure 3. Scheme of Research Process

3 RESULTS AND DISCUSSIONS

3.1 Phytochemical Screening

The results of the phytochemical screening of Citrus

hystrix leaf extract have been presented in the

following table.

Table 2. Phytochemical Screening Results of Citrus hystrix

leaf extract

Based on the results of the phytochemical

screening of kaffir lime leaf extract (Citrus hystrix) to

the six secondary metabolites as seen in Table 2, it

was found that the Kaffir lime leaf extract has a

chemical content consisting of flavonoids with Mg +

Amyl Alcohol + HClp powder reagent, glycosides

with Molish + reagent. H2SO4, Tannins with FeCl3

reagent, and triterpenoid / steroid with Lieberman-

Bourchat reagent, while secondary metabolites

Collection of Citrus hystrix leaf samples

Tools and materials preparation, and

Production of citrus hystrix leaf water

Preparation of the extract concentration (which

has been evaporated by ethanol) is then divided into

several concentrations

Phytochemi

cal

Screening

Manufact

ure of

simplicia

The tool is

sterilized at

121º C for 15

minutes.

The citrus hystrix leaves extract was made by

maserasi method.

Manufacture of Potato Dextrose Agar

The extract of citrus hystrix leaves using

maceration Ovale method

Pityrosporum Ovale Fungal Diffusion Test.

The hardened PDA media was scratched

evenly across the surface of the media using a

cotton swab containing the Pityrosporum Ovale

fungus suspension.

the zone of inhibition is measured using a

caliper.

An Experiment for Extracted Citrus Hystrix Leaf Effectiveness on Pityrosporum Ovale Fungi Growth

293

Alkaloid with Dragendroff Bouchardat Meyer

reagent, Saponin secondary metabolites with hot

water / shaken reagent were not found in the

phytochemical screening results of Citrus hystrix leaf

extract.

3.2 Inhibition Zone Diameter of Citrus

Hystrix Leaf Extract on

Pityrosporum Ovale Fungus

Growth

The effectiveness of citrus hystrix leaf extract to

inhibit the growth of Pityrosporum ovale fungus

using disc diffusion was shown by the presence of an

inhibitory or clear zone around the disc paper.

Inhibition zone was measured using a caliper. The

results of the study using a concentration of 25%,

50%, 75%, positive control and negative control can

be seen in the Figure 4:

Figure 4: Inhibition zone diameter of citrus hystrix leaf

extract on the growth of pityrosporum ovale fungus.

Table 3: Diameter of the Inhibition Zone of Pityrosporum

ovale

Concentration

Inhibition zone diameter

(mm)

Petri

Mean

25%

13,9

13,5

15,2

14,20

50%

15,3

14,8

16,1

15,40

75%

15,2

17,4

16,20

Mean

15,07

14,50

16,23

From Table 3, it is found that the citrus hystrix leaf

extract with the three concentrations, 25%

concentration, 50% concentration and 75%

concentration with 3 repetitions of petri 1, petri 2 and

petri 3. found that the concentration of 75% had an

increase in zone diameter. inhibition, the highest

diameter of the inhibition zone was at a concentration

of 75%, namely 16.20 mm, while the diameter of the

lowest inhibition zone was at a concentration of 25%,

namely 14.20 mm. The results of the inhibition zone

diameter are displayed in graphical form in the Figure

Figure 5: Graph of inhibition zone diameter of citrus hystrix

leaf extract on the growth of pityrosporum ovale fungus.

Figure 6: Average curve of inhibition zone of Citrus hystrix

leaf extract on the growth of the fungus Pityrosporum ovale

Based on the curve in Figure 6, it can be seen that at

a concentration of 25% the average diameter of the

inhibition zone is 14.20 mm, then there is an increase

in the average diameter of the inhibition zone at a

concentration of 50%, namely 15.40 mm, and at a

concentration of 75% an increase of 16, 20 mm. The

positive control inhibition zone diameter was 18.35

mm and the negative control inhibition zone diameter

was 0 mm.

4 DISCUSSIONS

Based on the results of the study, the leaf extract of

citrus hystrix (Citrus Hystrix) has antifungal

effectiveness against the growth of pityrosporum

ovale as indicated by the formation of an inhibitory

zone or clear zone around the disc paper. Then the

diameter of the zone of inhibition is measured using

a caliper to determine the amount of antifungal

power. The concentrations used were 25%, 50% and

75%. The results of the study based on the

Greenwood inhibition zone classification showed that

the leaf extract of citrus hystrix (citrus hysrix) with a

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concentration of 25%, 50% and 75% had antifungal

effectiveness against the growth of pityrosporum

ovale. With an average diameter of the inhibition

zone of 14.20 mm, 15.40 mm and 16.20 mm. whereas

in the positive control using ketoconazole 2% had a

stronger effectiveness in inhibiting the fungus

pityropsporum ovale with an average inhibition zone

diameter of 18.35 mm. for negative control using

aquadest did not have an inhibition zone in

pityrosporum ovale.

From the results of phytochemical screening, the

leaf extract of citrus hystrix (Citrus Hystrix) contains

flavonoids, tannins, glycosides and triterpenes /

steroids .. If tannins in low concentrations can inhibit

germ growth, while at high concentrations, tannins

work as an antimicrobial by coagulating or clumps of

bacterial protoplasm, so that it forms a stable bond

with the bacterial protein and in the digestive tract,

tannins are known to be able to eliminate toxins.

Whereas flavonoids have antifungal, antiviral and

antibacterial activity. Several studies have examined

the relationship between flavonoid structure and

antibacterial activity. Flavonoids can inhibit the

function of the cytoplasmic membrane and inhibit

energy metabolism. (Romawati, C et.al. 2017)

The ethanol extract of citrus hystrix leaves has a

larvicidal effect on Aedes aegypti larvae. The

concentration of ethanol extract of citrus hystrix

leaves is needed to kill 50% of the larvae population

of Aedes aegypti. (Santoso, L.M, 2015).

5 CONCLUSIONS

Based on the research results, it can be concluded that

the leaf extract of the citrus hystrix shows a clear zone

at all given concentrations. The 50% and 75%

treatments were the best treatments with an inhibitory

power of about 15.40 mm and 16.20 mm. The results

of phytochemical screening on the leaves of the citrus

hystrix obtained secondary metabolites such as

flavonoids, glycosides, tannins and triterpan.

ACKNOWLEDGMENT

The author would like to thank profusely to the Rector

of the Universitas Prima Indonesia, "Dr. Chrismis

Novalinda Ginting., M.Kes" who has supported the

research of novice lecturers with funding from the

Ministry of Research and Technology / the National

Research and Innovation Agency.

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