Antibacterial Activity of Methanol Extract of Calotropis gigantea

Flowers from Aceh

Kartini Hasballah

Department of Pharmacology, Faculty of Medicine,Universitas Syiah Kuala, Darussalam, Banda Aceh, Indonesia

Keywords: Antibacterial activity, Calotropis gigantea flower, Escherichia coli, Staphylocccus aureus

Abstract: The purpose of this study was to determine the antibacterial activity of methanol extract of Calotropis

gigantea flowers at various concentrations of gram-positive bacteria (Staphylocccus aureus) and gram-

negative bacteria (Escherichia coli). This study used a completely randomized design (CRD) consisting of 5

treatment groups and 2 control groups with 4 repetitions. The treatment group consisted of Calotropis

gigantea methanol extract with a concentration of 10%, 25%, 50%, 75% and 100% and the control group

consisted of negative control using 1% Sodium Carboxy Methyl Cellulose (CMC) and positive control

using ampicillin for S. aureus and chloramphenicol for E. coli bacteria. Antibacterial activity test was

carried out by disc diffusion method. Phytochemical test results showed that the methanol extract of

Calotropis gigantea flower contained alkaloids, steroids, terpenoids, flavonoids, saponins, tannins and

coumarin. Antibacterial test results of methanol extract of Calotropis gigantea flower on Staphylocccus

aureus and Escherichia coli showed that the methanol extract of Calotropis gigantea flowers had activity

against Staphylocccus aureus and Escherichia coli bacteria and increased concentration of methanol extract

of Calotropis gigantea flower followed by increased inhibition zone. Statistical analysis with ANOVA and

Duncan test at 95% Confidence Interval showed significant differences between treatment groups.

1 INTRODUCTION

Indonesia is a country rich in biodiversity, one of

which is vegetation. The role of plants has provided

many benefits for humans in the field of medicine,

one of which is the Calotropis gigantea plant (C.

gigantea). This plant is a shrub from the family

Apocynaceae which is often found in the regions of

Bangladesh, Burma, China, India, Indonesia,

Malaysia, Pakistan, Philippines, Thailand and Sri

Lanka (Sarkar et al., 2014). Some parts of this plant

such as leaves, stems, roots and flowers are

traditionally used to treat various diseases such as

tumors, fever, rheumatism, digestive disorders,

coughs, flu, asthma, nausea and diarrhea (Patel et al.,

2004). Ethnobotany part of the root of this plant is

efficacious to increase the flow of bile to the

intestine, healing skin infections, intestinal worms,

coughing asthma, bronchitis and dyspepsia (Kumar

et al., 2011).

A report stated that in India these plant roots are

used for sedatives, anticonvulsants, fever, laxatives

and deworming drugs (Argal et al., 2006). Plant

roots of C. procera which is a genus with the plant

C. gigantea, can treat colds and coughs, syphilis and

elephantiasis, can also function as anti-

inflammatory, analgesic, antimalarial and

antimicrobial (Kumar, 2009). Generally plants that

are in one genus such as C. gigantea and C. procera

contain the same secondary metabolites (Wink,

2010). Methanol extract of biduri plant roots caused

death in Aedes aegypti larvae (Zanaria et al., 2012).

Ethanolic extract of C. gigantea plant roots can heal

wounds faster in mice and this is in accordance with

the traditional use of C. gigantea root plants in India,

namely for wound healing (Deshmukh et al., 2009).

The methanol extract of this plant was active as an

insecticide against the larvae of Tribolium

castaneum (red flour beetle) (Alam et al., 2009).

gigantea flower extract has the potential as an

antimicrobial agent in the presence of terpenes,

where the activity of the extract was seen from the

Muller Hinton agar medium inhibition zone against

Pseudomonas aeruginosa, E. coli, and Tinea capitis

(Patil, 2012). Based on phytochemical analysis of

196

Hasballah, K.

Antibacterial Activity of Methanol Extract of Calotropis gigantea Flowers from Aceh.

DOI: 10.5220/0008788801960200

In Proceedings of the 2nd Syiah Kuala International Conference on Medicine and Health Sciences (SKIC-MHS 2018), pages 196-200

ISBN: 978-989-758-438-1

bioactive compounds in vitro in C. gigantea acts as

an antibacterial against many bacteria such as B.

cereus, B. subtilis, E. coli, K. pneumonia, S. aureus,

S typhii, Micrococcus luteus in all alkaloid extracts,

cardiac glycosides, tannins, saponins, flavonoids,

steroids, terpenoids sugar and resins (Seniya et al.,

2011). C. gigantea latex extract shown very good

fungicidal effects thus indicating that latex can be a

useful source for the development of new antifungal

agents against pathogenic fungi (Saratha &

Subramanian, 2010). Thus, the researcher were

interested in conducting an antibacterial activity test

from the methanol extract of Calotropis gigantea

flowers originating from Aceh against gram-positive

bacteria Staphylocccus aureus and gram-negative

bacteria Escherichia coli.

2 METHODS

2.1 Collection of C. gigantea Flowers

Calotropis gigantea flowers was collected from

Kayee Jatoe Cubo village dengan ketinggian 150 m

diatas permukaan laut, Kecamatan Bandar Baru,

Kabupaten Pidie Jaya during October 2017. The

plant was identified in the Herbarium Laboratory,

Department of Biology, Faculty of Mathematics and

Natural Sciences, Syiah Kuala University (Reg. No

of the certificate: No. 943/UN11.1.28.1/DT/2017).

The collected plant parts (flowers) were separated

from undesirable materials. The flowers were dried

in open air under shade for one week.

2.2 Tools and Materials

The tools used for this study were autoclave,

incubator, refrigerator, analytic scale, blender,

vacuum rotary evaporator (Heidolph), vortex,

spectrophotometer, ruler, petri dishes, test plates,

glass ware, micropipets, swab, scissor, label paper

and gloves.

The materials used in this study were methanol,

Calotropis gigantea flowers, gram positive bacteria

(Staphylocccus aureus) and gram negative bacteria

(Escherichia coli) obtained from the Aceh

Provincial Health Laboratory, blank discs, ampicillin

and chloramphenicol discs, 96% alcohol, aluminum

foil, Wagner, Mayer, and Dragendorf reagent,

magnesium powder, chloroform, sulfuric acid,

Lieberman-Burchard reagent, sterile aquadest,

Nutrient Agar (NA), Muller Hinton Agar (MHA)

media, sterile stick cotton.

2.3 Extraction Procedure

Calotropis gigantea flowers which have been dried

as much as 2 kg are smoothed into a blender until

they become powder then macerated with methanol

for 3x24 hours. The maserate obtained was

concentrated with a vacuum rotary evaporator at a

temperature of 50-60

C until a concentrated extract

was obtained. Calotropis gigantea flowers extract

100% pure diluted in various concentrations,

namely: 10, 25, 50, 75 and 100%.

2.4 Phytochemical Analysis

The fresh flowers and concentrated methanol

extracts have been conducted a phytochemical test

as Harborne phytochemical method to see whether

they contain secondary metabolites (Table 1)

(Harborne, 2006).

2.5 Antibacterial Activity Test

Antibacterial activity test was carried out by Kirby-

Bauer disc diffusion method. This test uses Muller

Hinton Agar (MHA) media with a depth of 4 mm

(25 ml of MHA media for 100 mm petri dishes and

60 ml for 150 mm petri dishes) (Hood et al., 2010).

The bacterial suspension that will be used for

antibacterial power tests is measured in density

using a spectrophotometer (λ = 625 nm and

absorbance 0.08 - 0.13) to obtain the bacterial

density standard at 1-2 x 10

CFU/ml as stated by

European Committee on Antimicrobial

Susceptibility Testing (Kahlmeter, 2006).

Then the

bacteria were inoculated into the MHA media by

dipping sterile stick cotton into the inoculum and

then rubbing it on the entire surface of the MHA

media three times by rotating the cup at an angle of

C after each application so that the flat media

surface was filled with bacterial suspension. Control

discs, antibiotic discs (ampicillin or

chloramphenicol) and discs containing various

concentrations of samples were placed on the

inoculation media using sterile tweezers and pressed

slowly and incubated at 37

C for 24 hours. After

incubation, the diameter of the inhibitory zone in the

form of clear zone is measured using a ruler in

millimeters (mm) and compared with the inhibition

power classification (Morales et al., 2003).

Antibacterial Activity of Methanol Extract of Calotropis gigantea Flowers from Aceh

197

2.6 Data Analysis

The data obtained in this study, in the form of

inhibitory zone diameter growth of Staphylocccus

aureus gram-positive bacteria and Escherichia coli

gram-negative bacteria in various concentrations of

methanol extract of Calotropis gigantea flowers

tested for normality with Kolmogorov-Smirnov test

and homogenity test with Lavene test. Then

ANOVA test was conducted at 95% Confident

interval (CI) and continued with Duncan test.

3 RESULTS AND DISCUSSION

3.1 Total Yield Extracts and Its Color

Extraction 2 kg flowers of C. gigantea produced

1.97% methanol extract with greenish yellow color.

3.2 Phytochemical Constituents

C. gigantea flowers and methanol extract of C.

gigantea flowers contain many secondary

metabolites. Phytochemical test results for C.

gigantea fresh flowers contain alkaloids, streroids,

saponins, phenols, flavonoids, tannins and coumarin.

whereas the methanol extract of C. gigantea flowers

obtained secondary metabolites of alkaloids,

steroids, phenols, flavonoids, tannins and coumarin

(Table 2). This shows that methanol as a polar

solvent can attract many compounds which have the

potential to be used as drugs.

Table 1. Phytochemical Test of Flower and Methanol Extract of C. gigantea Flower

Chemical

content

Reagent

Phytochemical Test

Results of C. gigantea

flowers

Phytochemical Test

Results of Methanol

Extract of C. gigantea

flowers

Information

Alkaloi

Maye

+ + White sediment

+ + Chocolate sediment

Dragendorff + + Red sediment

Steroid

Liebermann-

Burchard Test

+ + Green or blue

Terpenoid

Liebermann-

Burchard Test

- - Red

Saponin Shuffle + - Foaming

Phenol FeCl

5% + + Dee

Blue or Blac

Flavonoi

Mg dan HCl + + Re

Tannin Dilute HNO

+ +

Change from

Reddish to Yellow

Coumarin Ammonia Steam + +

Presence of

fluorescence

+ present - absent

The presence of tannins shows the ability of this

plant to play an important role as an antidiarrheal

and antihemorhagic agent (Asquith & Butler, 1986).

Phenol compounds are known to have several

subclasses, namely simple phenols, phenolic acids,

quinones, flavonoids, flavones, flavonols and

tannins (Cowan, 1999). Phenol compounds are

known to have antibacterial properties. Mechanism

phenol compounds as antibacterial substances are by

penetrating the cell wall and into the cytoplasm and

then precipitate the protein in the microbial cell.

Phenol compounds can also denaturize enzymes that

function in germination of spores or affect the amino

acids responsible for the germination process. Large

molecular phenolic compounds are able to activate

essential enzymes in microbial cells even in low

concentrations. Phenol compounds can break the

peptidoglycan bond when breaking through the cell

wall. This will cause cell lysis (Hariyati, 2010).

The antimicrobial activity of the methanol

extract of C. gigantea flowers studied in this study

was assessed qualitatively by measuring the

diameter of the inhibition zone.

SKIC-MHS 2018 - The 2nd Syiah Kuala International Conference on Medicine and Health Sciences

198

Table 2. Antibacterial activity test results of methanol extract of C. gigantea

Treatment

/ Control

Concentration (%) Inhibitor

Zone Diameter

(

)

Classification

based on Morales et al.

S. aureus E. coli

10 6 7 +

25 11 17 ++

50 18 19 ++

75 23 22 +++

100 28 25 +++

CMC 1% 0 0 -

Antibiotics 24

(Ampicillin)

(Chloramphenicol)

+++

Information:

: Methanol Extract of C. gigantea flowers with 10% concentration

: Methanol Extract of C. gigantea flowers with 25% concentration

: Methanol Extract of C. gigantea flowers with 50% concentration

: Methanol Extract of C. gigantea flowers with 75 % concentration

: Methanol Extract of C. gigantea flowers with 100% concentration

: CMC 1% as negative control

: Antibiotics as positive controls (ampicillin for S. aureus; chloramphenicol for E. coli)

3.3 Antibacterial Activity

The antimicrobial activity of the methanol

extract of C. gigantea flowers studied in this

study was assessed qualitatively by measuring

the diameter of the inhibition zone. To

determine the zone of inhibition, Gram-

positive, Gram-negative were taken as standard

antibiotics (ampicillin, chloramphenicol) for

comparison of results. The results of this study

revealed that the extracts of the plants tested

had potential antimicrobial activity against

Gram-positive and Gram-negative

microorganisms.

4 CONCLUSIONS

Methanol extract of C. gigantea has

antibacterial activity against S. aureus and E.

coli bacteria and also increasing the

concentration of methanol extract of C.

gigantea flowers followed by an increase in

inhibitory zones.

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