Phytochemical analysis of Binahong (Anredera Cordifolia) leaves extract to inhibit In

Vitro growth of Aeromonas Hydrophila

Mohammad Basyuni, Prita Yulianti Anasta Br Ginting, and Indra Lesmana

Citation: AIP Conference Proceedings 1904, 020072 (2017);

View online: https://doi.org/10.1063/1.5011929
View Table of Contents: http://aip.scitation.org/toc/apc/1904/1
Published by the American Institute of Physics

Articles you may be interested in

Dyes removal using activated carbon from palm oil waste with digital image colorimetry quantification
AIP Conference Proceedings 1904, 020066 (2017); 10.1063/1.5011923

Optimization of chromatographic conditions for determination of aflatoxin B1, B2, G1 and G2 by using liquid
chromatography-mass Spectrometry
AIP Conference Proceedings 1904, 020071 (2017); 10.1063/1.5011928

Effect of citronella essential oil fractions as oil phase on emulsion stability

AIP Conference Proceedings 1904, 020070 (2017); 10.1063/1.5011927

Kinetic studies of adsorption in the bioethanol dehydration using polyvinyl alcohol, zeolite and activated carbon
as adsorbent
AIP Conference Proceedings 1904, 020076 (2017); 10.1063/1.5011933

Hydrodeoxygenation of bio-oil using different mesoporous supports of NiMo catalysts

AIP Conference Proceedings 1904, 020078 (2017); 10.1063/1.5011935

Disinfection of Escherichia coli bacteria using hybrid method of ozonation and hydrodynamic cavitation with
orifice plate
AIP Conference Proceedings 1904, 020075 (2017); 10.1063/1.5011932
 Phytochemical Analysis of Binahong (Anredera Cordifolia)
Leaves Extract to Inhibit In Vitro Growth of Aeromonas
Hydrophila
Mohammad Basyuni1a), Prita Yulianti Anasta Br Ginting2 and Indra Lesmana2
1
Program Studi Kehutanan, Fakultas Kehutanan, Universitas Sumatera Utara, Jl. Tri Dharma Ujung No. 1 Medan,
Sumatera Utara 20155, Indonesia
2
Program Studi Sumberdaya Perairan, Fakultas Pertanian, Universitas Sumatera Utara, Medan 20155, Indonesia
a)
Corresponding author: m.basyuni@usu.ac.id

Abstract. Binahong (Anredera cordifolia) is one of the medicinal plants commonly used to treat the disease of living
organisms. The secondary metabolite of A. cordifolia leaves has been shown antibacterial activity. This study aimed to
investigate the secondary metabolite of A. cordifolia leaves showing antibacterial and analysis the effectiveness of
antibacterial to inhibit the growth of bacteria Aeromonas hydrophila. A paper disc soaked in a solution of A. cordifolia
leaves extract was used to test in vitro at a concentration of 0% (w/v), 0.2%, 0.4%, 0.6%, 0.8%, and positive control of
antibiotic (oxytetracycline), respectively. The extracts then placed on a tryptone soy agar (TSA) medium containing
bacteria A. hydrophila and incubated at 37 ºC for 24 hours. In vitro test showed that A. cordifolia leaves extract inhibited
the growth of bacteria A. hydrophila with an inhibition area around the paper disc. The inhibition growth of A. hydrophila
increased with the increasing of extract concentration. Bacterial growth was inhibited in the diameter zone of A. hydrophila
under different levels of the extracts were 0 mm (0 % negative control ), 8.4 mm (0.2 %), 9.4 mm (0.4 %), 10.5 mm (0.6
%), 11.9 mm (0.8 %), 27.5 mm (positive control), respectively. Phytochemical screening of A. cordifolia leaves extract
indicated that the extracts contained flavonoid, phenol, saponin, alkaloid, triterpenoid, and β-sitosterol. Our in vitro study
demonstrated the inhibition growth of A. hydrophila that caused the disease of motile Aeromonas septicemia (MAS).

INTRODUCTION
The most frequent problem in the process of freshwater fish farming was the occurrence of disease attacks [1].
One of the few conditions that often cause freshwater fish death is motile Aeromonas septicemia (MAS). MAS disease
was mainly caused by a bacterial infection of Aeromonas hydrophila [2]. The efforts to control MAS disease until
now rely on antibiotics in fish farming [2]. However, the utility of antibiotics for long-term, uncontrolled, overused,
and inappropriate doses may have adverse impacts on environments [3]. This impact is not only lead to new strains of
bacteria resistant to antibiotics that may endanger humans and but also pollute the aquatic environment, even affecting
people's health because of chemical residues of antibiotics in fishery products consumed [3].
A new approach to look for the alternative method of controlling MAS disease may become of an urgent concern
in a decade [2]. The uses of the natural product to control diseases that attack the fresh water fish are required in
replacement of chemicals to reduce environmental pollution. Nonetheless, a few studies on the controlling from plant
material were reported. Binahong (Anredera cordifolia) is one of the medicinal plants commonly used to treat the
disease of living organisms. It has been shown that A. cordifolia has a broad range of content of secondary metabolites
that may be useful in the treatment of illness [4]. To get more insight into the biological function of the medicinal
plant, A. cordifolia leaves were tested for the MAS disease, using in vitro experiment under varying concentration of
leaves extract on the growth of bacteria Aeromonas hydrophila. This study aimed to examine the secondary metabolite
of A. cordifolia leaves showing antibacterial and analysis the effectiveness of antibacterial to inhibit the growth of
bacteria A. hydrophila.

Proceedings of the 3rd International Symposium on Applied Chemistry 2017

AIP Conf. Proc. 1904, 020072-1–020072-5; https://doi.org/10.1063/1.5011929
Published by AIP Publishing. 978-0-7354-1594-2/$30.00

020072-1
 MATERIALS AND METHOD
The Binahong leaves (approximately 400 g) were obtained from home garden in Binjai, North Sumatra. Leaves
were washed and air drained for five days. Dried leaves were ground using blender in homogeneous powder and stored
in a sealed glass container and not exposed to direct sunlight. 50 g of powder was dissolved in 500 ml of 96% ethanol
covering with aluminum foil and left for two days with stirring occasionally. After two days, the samples filtered with
a cotton swab. The filtrate was evaporated to obtain a concentrated binahong leaf extract. Extracts were stored in
sealed glass bottles [5].
Phytochemical screening of steroids/terpenoids was carried out as previously described [6]. 2 ml sample extract
was inserted into a test tube, to test steroids/terpenoids, the extract was added to 2 drops of reagent Liebermann-
Bouchard. The blue color formed purple/blue, green color showed steroids/terpenoids. Terpenoid was tested using
thin layer chromatography (TLC) with a sample then dripped into TLC plate sprayed with 1 % CeSO 4 reagent and
heated on a hot plate. The color changes observed and compared with the standard of lupeol and β-sitosterol [7].
Phytochemical screening of phenolic and flavonoid was performed as previously reported [5]. 1 ml of sample
extract was inserted into a test tube to test phenolic extracts by 1 % FeCl3. A black color indicates the presence of
phenolic compounds. The flavonoid compounds separated by employing the dried leaf powder (crude drug), added 5
ml of ethyl acetate, shaken carefully and stood for a while, before adding 1 % FeCl 3 reagent. The color changes to
black indicated flavonoids.
Analysis of A. hydrophila bacteria using gram staining, motility test, oxidase test, O/F (oxidative/fermentative)
test, and Rimmler-Shoots (RS) test as described previously [8]. These extracts were used to verify anti-bacterial with
the concentration of 0 % (negative control), 0.2 %, 0.4 %, 0.6 %, 0.8 % and oxytetracycline as positive control.
Antibacterial tested by the disk diffusion method using paper discs as previously reported [8]. An aqueous
suspension of bacteria spread on TSA medium and then allowed to stand for a while. Paper discs soaked with different
concentrations of the test solution for 15 minutes placed on TSA media that previously had been spread bacterial
suspension solution was then incubated at 37 °C for 24 hr [8]. The bacterial growth described by comparing the
inhibition zone among the treatments.

RESULTS AND DISCUSSION

Phytochemical screening showed that the extract contains secondary metabolites namely phenolic, flavonoids,
terpenoid/steroid (triterpenoid and β-sitosterol), alkaloids, and saponins as displayed in Figure 1 and Table 1. Based
on phytochemical screening of phenolic (Table 1) obtained positive reactions to FeCl3 reagents characterized by a
black color. The FeCl3 reagent is a specific reagent for compounds derived from phenolic and flavonoids which are
derivatives of phenol (Table 1). The presence of flavonoids with 1% FeCl3 reagent indicated the change of color to
black.

FIGURE 1. TLC chromatogram of the binahong leaf extract showed that triterpenoid (lupeol) and β-sitosterol corresponded to
the standard accordingly

Figure 1 showed the results of steroid/terpenoid obtained positive results on Liebermann-Bouchard reagents
characterized by a blue-greenish coloration and positive reactions to Liebermann-Bouchard reagents showed the
presence of steroid/terpenoid compounds. Furthermore, the extract was tested using TLC method (1% CeSO4) reagent
to include the steroids and terpenoids group. The obtained results are positive triterpenoids and β-sitosterol
characterized by the color change in the extract that resembles the standard colors of triterpenoids and β-sitosterol as
depicted in Figure 1.

020072-2
 Table 1 displays phytochemical screening of alkaloid showed negative results (no change in color) on Bouchardat
reagent, Wagner reagent, Mayer reagent, but reacted positively to Dragendroff reagent marked by sediment (data not
shown). The phytochemical screening of saponin (Table 1) showed positive results indicated by stable froth for 10
min with height 1-10 cm and foam not lost if added one drop (0.05 ml) 2N HCl.
TABLE 1. Phytochemical screening of secondary metabolites in the A. cordifolia leaf extracts
Secondary metabolite Solvents Signal
Phenolic FeCl3 +++
Steroid/Terpenoid Libermann-Bouchard +++
Lupeol (TLC) +
β-sitosterol (TLC) +
Alkaloid Bouchardat -
Wagner -
Mayer -
Dragendroff ++
Saponin Aquades-HCl +++
+ : Change color (positive reaction) lasts a long time on the first few drops of reagent additions.
++ : Changing color (positive reaction) takes place slowly at first drops of reagent addition (estimated 1 drop = 0.05 ml).
+++ : Direct changes color (positive reaction) with the addition of the first reagent drops (approximately 1 drop = 0.05 ml).

Analysis of bacteria criteria on A. hydrophila demonstrated that pure cultures of bacteria were A. hydrophila
bacterium (Table 2). Antibacterial testing performed disk diffusion method using paper discs with a diameter of 6
mm. Observations made after an incubation period of 24 hours and results were presented in Table 3 and Figure 3.
Gram staining showed that the characteristic of gram negative bacteria characterized by red/pink bacteria cell (Table
2). This result indicated the gram-negative bacteria release the first color purple of crystal violet and bind to a second
dye of safranin [3].
TABLE 2. Analysis of bacteria in A. hydrophila
Test Result
Gram stain Gram-negative, short rod shape
Motility test Motile
Oxidase test Oxidase positive
Oxidative/Fermentative Oxidative/Fermentative positive
test
Rimmler-Shoots test Rimmler-Shoots positive

The results of in vitro of binahong leaf extract to A. hydrophila bacteria depicted that the extract of the binahong leaf
could inhibit the growth of A. hydrophila bacteria that causing fish disease. The results of this research were parallel
with natural ingredients of binahong leaves can be used as an alternative in the treatment of fish because the leaves of
binahong can inhibit the growth of bacteria that cause fish diseases in vitro [2].

FIGURE 2. Oxidase test showed a positive reaction to oxidase marked with a blue-purple color of the scratches on paper
oxidase

020072-3
 TABLE 3. Inhibition zone diameter measurement of binahong leaf extract on the growth of bacteria A. hydrophila
Inhibition zone diameter (mm)
Concentration test (%) Mean inhibition zone diameter (mm)
singlicate duplicate triplicate
Water (negative control) 0 0 0 0
Oxytetracycline (positive control) 27.4 27.6 27.4 27.5
0.2 8.1 8.6 8.5 8.4
0.4 9.2 9.6 9.4 9.4
0.6 10.3 10.5 10.7 10.5
0.8 12.4 11.6 11.6 11.9

Measurement of inhibition zone diameter showed that binahong leaves extract against bacterial A. hydrophila in
the paper disc without binahong leaf extract were not inhibited. This finding as evidenced by clear zone diameter/0
mm inhibition. Distilled water does not have the active substances to inhibit the bacteria [9]. Paper discs containing
the antibiotics of oxytetracycline (positive control) showed a broad zone of inhibition. Antibiotic of oxytetracycline
is part of the class of tetracyclines. Tetracycline is a broad-spectrum antibiotic inhibit protein synthesis [4]. Paper discs
containing a different concentration of juice binahong leaves showed variable inhibition zone diameter (Figure 3).
Smallest inhibition zone diameter was obtained at a concentration of 0.2 % binahong leaf extract (8.4 mm) and the
largest one 0.8 % (11.9 mm). Paper discs applied was different levels of the solution compared to paper discs
containing distilled water showed binahong leaf extract inhibited the growth of bacteria and affected secondary
metabolites, such as flavonoids, phenolics, alkaloids, saponins, triterpenoids, and β-sitosterol. Flavonoids were
reported as an antibiotic to interfere the function of microorganisms such as bacteria [4]. Flavonoids have tested in
vitro is more useful as an antimicrobial agent against a variety of microorganisms [8]. Saponin has reported as an
antimicrobial [9]. Alkaloid as the class of heterocyclic nitrogen compounds was reported to have antibacterial
properties [9]. The presence of phenolic compounds therefore also affected the inhibition of bacteria [8]. Phenolic
employed by protein denaturation and cell damage bacterial cell walls to stunt the growth of bacteria [9].

FIGURE 3. Inhibition zone leaf extract binahong against the growth of bacteria A. hydrophila.

CONCLUSION
Phytochemical screening of A. cordifolia leaves extract containing secondary metabolites namely phenolic,
flavonoids, triterpenoids, β-sitosterol, and saponins. In vitro test demonstrated that A. cordifolia leaves extract
inhibited the growth of bacteria A. hydrophila with an inhibition area around the paper disc. Our finding confirmed
the promising of A. cordifolia leaves extract to inhibit the growth of bacteria A. hydrophila.

ACKNOWLEDGMENTS
The authors are grateful the Ministry of Research, Technology and Higher Education through Excellent Research
for Higher Education 2017 (No. 003/SP2H/LT/DRPM/IV/2017).

REFERENCES
1. K. D. Lafferty, C. D. Harvell, J. M. Conrad, C. S. Friedman, M. L. Kent, A. M. Kuris, E. N. Powell, D. Rondeau
and S. M. Saksida, Annu. Rev. Mar. Sci. 7, 471–496 (2015).
2. H. Y. Done, A. K. Venkatesan and R. U. Halden, AAPS J. 17, 513–524 (2015)

020072-4
3. K. Pulkkinen, L.-R. Suomalainen, A. F. Read, D. Ebert, P. Rintamäki and E. T. Valtonen, Proc. Biol. Sci. 277,
593–600 (2010).
4. L. F. Souza, I. B. de Barros, E. Mancini, L. De Martino, E. Scandolera and V. De Feo, Nat. Prod. Commun. 9,
1003–1006 (2015).
5. R. Nagappan, Asian Pac. J. Trop. Biomed. 2, 707–711 (2012).
6. M. Basyuni, H. Oku, S. Baba, K. Takara and H. Iwasaki, J. Oceanography 63, 601–607 (2007)
7. M. Basyuni, S. Baba, Y. Kinjo, L. A. P. Putri, L. Hakim and H. Oku, J. Plant Physiol. 169, 1903–1908 (2012).
8. S. U. T. Pratiwi, E. L. Lagendijk, T. Hertiani, S. d. Weert, C. A. M. J. J. V. D. Hondel, J. Horticulture 2, 119
(2015). doi: 10.4172/2376-0354.1000119.
9. J. Zhang, X. Rui, L. Wang, Y. Guan, X. Sun and M. Dong, Food Control 42,125–131 (2014).

020072-5