RESEARCH ARTICLE | SEPTEMBER 06 2019

Antimicrobial activity of Acorus calamus L. rhizome extract

and its total flavonoid and phenolic contents 
Wiwik Susanah Rita  ; I. Made Dira Swantara; Gusti Ayu Primandani Utami

AIP Conf. Proc. 2155, 020054 (2019)

https://doi.org/10.1063/1.5125558

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 Antimicrobial Activity of Acorus calamus L. Rhizome
Extract and Its Total Flavonoid and Phenolic Contents
Wiwik Susanah Rita1,a), I Made Dira Swantara2,b) and Gusti Ayu Primandani
Utami2,c)
1
Chemistry Department, Faculty of Math. And Natural Science, Udayana University, Bali, Indonesia.
2
Magister of Applied Chemistry Department, Faculty of Math. And Natural Science, Udayana University, Bali,
Indonesia.
a)
Corresponding author: susanah.rita@unud.ac.id
b)
dira_swantara@unud.ac.id
c)
primandariutami@gmail.com

Abstract. Acorus calamus L. rhizome has potential activity as an antimicrobial agent. This study aims to evaluate the
antimicrobial activity of the rhizome extracts and its total flavonoid and phenolic contents. Extraction was carried out by

07 September 2023 11:30:32

using ethanol at room temperature followed by fractionation into n-hexane, ethyl acetate, and n-butanol. Antimicrobial
activity towards Escherichia coli, Staphylococcus aureus, and Candida albicans were evaluated by agar diffusion
method. Total flavonoid and phenolic contents were determined by UV-Vis Spectrophotometer using the standard of
quercetin and gallic acid, respectively. The A. calamus rhizome ethyl acetate fraction strongly inhibited Escherichia coli,
Staphylococcus aureus and Candida albicans, followed by n-butanol and n-hexane fractions, respectively. There was no
inhibition of water fraction towards all the microbes. Total flavonoid contents of ethyl acetate, butanol, n-hexane, and
water fractions were successively 2978.34, 399.07, 142, 23, and 14.80 mg QE/100g, while total phenolic contents of
those fractions were 1820.51, 329.71, 237.81, and 74.36 mg GAE/100g, respectively. The results show that there is a
positive correlation between antimicrobial activity and its total flavonoid and phenolic contents.

INTRODUCTION
Infectious diseases are a great burden on many societies. The disease may caused by bacteria, fungi, viruses,
protozoa, and multicellular parasites. The infections could spread through skin contact, inhalation, ingestion,
transfusion, unprotected sex, and others [1]. The microbes such as Escherichia coli, Staphylococcus aureus and
Candida albicans are a major human pathogen that causes a wide range of clinical infections. Generally, the
pathogens are controlled by Synthetic drugs. However, the drugs are not only expensive and inadequate for the
treatment of the diseases but also often have side-effects [2]. The drug side-effects, or adverse drug reactions, have
become a major healthcare concern [3]. As an illustration to the extent of this problem, serious drug side-effects are
estimated to be the fourth leading cause of death in the US, resulting in 100,000 deaths per year [4]. Friedman et al.
stated that the spread of resistance in healthcare settings and the community threatens the availability of antibiotic
therapy [5]. The antibiotic resistance is responsible for the loss of the effectiveness of antimicrobial agents.
Therefore, the development of a new medicine is required to overcome this problem. Various specific plants, as
sources of medicinal compounds have continued to play a dominant role in the maintenance of human health.
Rahayu et al. [6] reported that the leaves of Sesbania grandiflora (L.) can be used as antiseptic, while the leaves and
fruits of Psidium guajava L can be applied to treat diarrhea. Ginger (Zingiber officionale Rosc.) was traditionally
used for stimulating mucous membranes which makes it effective as an appetite enhancer and also for the digestive
system. It is also used to cure headache and colic, the essential oils were used for antioxidant and antiseptic
properties [7]. Medicinal plants may offer a new source of antimicrobial agents [8]. Biologically, the active
compounds from natural sources have always been of great interest to scientists working on infectious diseases [2].

Proceedings of the 2nd International Conference on Biosciences and Medical Engineering (ICBME2019)
AIP Conf. Proc. 2155, 020054-1–020054-9; https://doi.org/10.1063/1.5125558
Published by AIP Publishing. 978-0-7354-1900-1/$30.00

020054-1
 A variety of plant species for infectious diseases treatment are traditionally used in some countries, especially
South East Asia. Gonelimali et al. [9] evaluated the antimicrobial potential of ethanol and water extracts of Hibiscus
sabdariffa, Syzygium aromaticum, Rosmarinus officinalis, and Thymus vulgaris on some food pathogens and
spoilage microorganisms. Khan et al. [10] reported that ethanol extract of Azadiracta indica, Allium sativum, Cordia
dichotoma, Ocimum sanctum, Syzygium cumini, and Trigonella foenum grecum were effective against Candida
isolates with inhibition zone ranging from 10- 18mm in diameter, and the minimum inhibitory concentration (MIC)
was recorded ranging from 1.56-25mg/ml. The methanol extract of Microdesmis puberula exhibited antimicrobial
activity against both Gram-positive and Gram-negative bacteria with inhibition zones ranging from 12 to 16 mm
[11]. Padder et al. [12] investigated the bacterial activity of Vitex negundo, Duranta repens, Piper nigrum, and
Acorus calamus extracts. It was found that the plant extracts were effective against S. mutans and P. aeruginosa.
Acorus calamus L. demonstrates many important biological activities, particularly antimicrobial properties.
Susanah et al. [13] reported that the ethanol extract of A. calamus L. rhizome possessed antimicrobial activity
against Escherichia coli, Staphylococcus aureus, and Candida albicans. The MIC of the extracts against Escherichia
coli, Staphylococcus aureus and Candida albicans was 2, 3 and 3% respectively with the inhibition zone of 9.80,
9.50 and 8.67 mm. The ethanol and methanol extracts of the rhizome have the ability to inhibit the growth of E. coli,
S. epidermidis, and S. aureus, with similar effects [14]. The activities of A. calamus L. have also reported, such as
antifungal against Fusarium oxysporum f. sp. lycopersici [15], anticancer and antioxidants [16], antioxidant,
antimicrobial, and insecticidal activity [17], antibacterial, antifungal, Antiulcer and cytoprotective, anti-
inflammatory, anti-diabetic, anti-cellular and immunosuppressive, insecticidal, anti-diarrheal, antiviral and anti-
anginal, anti-rheumatitis, and anti-hepatotoxi activity [18].
Rahamoz-Haghighi et al. [14] revealed that ethanol extract of A. calamus contained phenyl propanoids,
monoterpenes, sesquiterpenes and D-asarone that possessed antibacterial activity. Essential oils isolated from A.
calamus rhizome potentially inhibited the growth of S. aureus and E. coli [19] and Candida albicans [20] with the
MIC of 0.4; 4.0; and 1% respectively, the oils consist of E-asarone (18,62 %), v- asarone (18,29 %), and euasarone
(26,84 %). The oils were also potentially active against Fusarium solani, a fungal pathogen on dragon fruits [21,

07 September 2023 11:30:32

22]. The oils strongly inhibited F. solani with the MIC of 2%. We have conducted phytochemical test of the ethanol
extract of A. calamus rhizome collected in Denpasar. It showed that the extract contained alkaloids, steroids,
triterpenoids, flavonoids, and polyphenols. Besides essential oils, the antimicrobial activity of A. calamus rhizomes
may be caused by the presence of flavonoids and polyphenols. Mahboubi et al.[23] stated that the antimicrobial
activity of the plant extracts has a positive correlation with their phenolic and flavonoid contents. The compounds
can be obtained through extraction using ethanol, followed by fractionation into n-hexane, ethyl acetate, and n-
butanol. Therefore, it is necessary to evaluate the antimicrobial activity of the rhizome fractions and their total
flavonoid and phenolic contents, so that it can be known the relationship between antibacterial activity and those of
content.

MATERIAL AND METHODS

Plant Material
A. calamus L. rhizomes were collected around Denpasar Bali.Classification of the plants were done at LIPI-UPT
Center for Plant Conservation Botanical Garden "Eka Karya" Bali. The rhizomes were cleaned, cut, and dried at
room temperature for 15 days. Then, they were milled into powder and stored for the next process.

Microbial Agents
Microbial used for antimicrobial assay were E. coli (Gram negative bacteria) and S. aureus (Gram positive
bacteria) and C. albicans (fungal pathogen). These microorganisms were obtained from culture collection from the
Laboratory of Microbiology, Central Public Hospital Clinic of Sanglah Denpasar. The isolates were purified by
touching the surface of the microbe population (using osse needle) and scratched into a new media. The isolates
were then maintained at 4°C until further use.

020054-2
 Extraction
A. calamus rhizome powder was extracted with ethanol 96% for 24 h at room temperature (25°C). The extract
was filtered and then evaporated using a rotary vacuum evaporator. The ethanol extract was then dissolved in
ethanol: water (3:7), ethanol was evaporated to obtain water extract. Water extract was fractionated into solvents
from non-polar solvent to polar solvent. They were n-hexane, ethyl acetate, and n-butanol respectively. The fractions
obtained were evaporated to get n-hexane, ethyl acetate, and n-butanol concentrated fractions, and then they were
stored at 4°C until further analysis.

Antimicrobial Activity Assay

Antimicrobial activity assay of all fractions was carried out by the well diffusion method. The concentration
applied to the assay was 20% with three repetitions. Nutrient agar media was used for the antibacterial assay, while
PDA (Potato Dextro Agar) was used for the antifungal assay. Petri dish containing 200 μl of the microbe suspension
(E. coli, S. aureus and C. albicans) was added 10 mL of media and allowed to solidify. The diffusion wells were
made using a cork borer, then each well was filled with 20 μl of the fraction and incubated at a temperature of 37°C
for 48 hours. The diameter of the clear zone showed the inhibition of the growth of the microbes [19]. Minimum
Inhibitory Concentration (MIC) was determined with the same methods using various concentrations (2, 4, 6, 8, 19,
and 15%)

Phytochemical Screening
Secondary metabolites in A. calamus rhizome were phytochemically screened using detection reagents of
compound groups, such as triterpenoids, steroids, alkaloids, flavonoids, phenolic compounds, and saponins. 1)
triterpenoid and steroid test: 1 mL of extract was added with 0.5 ml of acetic anhydride and then slowly added 2 ml

07 September 2023 11:30:32

of concentrated H2SO4 (Lieberman-Burchard Test), a red brown coloration was indicated the presence of
triterpenoids, while that a blue green coloration was indicated the presence of steroids; 2) alkaloid test: 1 ml of
extract was dissolved in HCl, then added with 2-3 drops of Mayer reagent. Identification of alkaloid was
demonstrated by the formation of a white precipitates; 3) flavonoid test: 0.5 ml extract was added with concentrated
HCl (Bate-Smith Test), and then heated for 15 minutes, a red coloration signified the presence of flavonoids; 4)
phenolic test: 1 mL of extract was added with 1% FeCl3. The formation of a green, red, purple, blue, or black
confirmed the presence of phenolics; 5) saponin test: 1 mL of extract was added with 2 mL of boiling water and
allowed to stand for 15 min, then shaken vigorously. The presence of saponins was confirmed by the formation of a
stable foam.

Determination of Total Phenolic and Flavonoid Contents

Total Flavonoid contents

Total flavonoid contents were determined by the aluminum chloride method according to Rita et al. [24] with
modification. A total of 53.8 mg of n-hexane, 50.9 mg of ethyl acetate, 55.4 mg of n-butanol, and 51.6 of water
fractions were dissolved in 96% ethanol to obtain a volume of 5 ml. Absorbance at 415 nm was recorded after 90
minutes of incubation. A series of standard quercetin solutions with various concentrations were also prepared and
the absorbance was recorded at a wavelength of 415 nm. The calibration curve of standard quercetin was made to
obtain the equation line of Y = ax + b. The total flavonoid contents were expressed as mg quercetin equivalents
(QE)/100 mg fraction.The total flavonoids can be determined by using the following formula:

C.V.F.10 -6
F1 u100% (1)
m
where F1 = total flavonoid contents, C = equality of quercetin(g/mL), V = total volume of fraction (mL), F = the
dilution factor and m = weight of sample (g).

020054-3
 Total Phenolic contents

Total phenols were assayed according to Rita et al. [24] with modification. A total of 121.80 mg of n-hexane,
52.30 mg of ethyl acetate, 103.90 mg of n-butanol, and 288.00 of water fractions were dissolved in 96% ethanol to
obtain a volume of 5 ml. 100 μL of Folin-Ciocalteu reagent and 800 μl of 5% sodium carbonate were added to the
solution. Absorbance at 760 nm was recorded after 90 minutes of incubation. A series of standard gallic acid with
various concentrations were also prepared and the absorbance was recorded at a wavelength of 760 nm. The
calibration curve of standard gallic acids was made to obtain the equation line of Y = ax + b. The total phenolic
contents were expressed as mg gallic acid equivalents (GAE) /100 g of the fraction. The total phenols can be
determined by using the following formula:

C.V .F .10 6
F2 u100% (2)
m
where F1 = total phenolic contents, C = equality of gallic acid (g/mL), V = total volume of extract (mL), F = the
dilution factor and m = weight of sample (g).

RESULTS AND DISCUSSION

Extraction
Extraction of 750 g A. calamus rhizome powder produced 139.5 g of ethanol extract. Fractionation of 80 g
concentrated ethanol extract of the rhizome with n-hexane, ethyl acetate and n-butanol produced n-hexane (4.3 g),
ethyl acetate (2.5 g), n-butanol (3.8 g), and water (10.6 g) fractions.

07 September 2023 11:30:32

Phytochemical Screening
Phytochemical screening was carried out to the crude (ethanol) extract and fraction resulted from fractionation.
The result of the screening can be shown in Table 1. Based on Table 1, it can be seen that the crude extract and all
fractions did not contain saponins. Ethanol extract contained all the secondary metabolites screened, except
saponins. Phenolic compounds contained in all the fractions, while flavonoids were in ethanol, ethyl acetate, n-
butanol, and water fractions. This result was in line with the study of Rahamoz-Haghighi et al. [14]. They revealed
that ethanol extract of A. calamus contained phenyl propanoids, monoterpenes, sesquiterpenes and D-asarone that
possessed antibacterial activity. Meanwhile, Mahboubi et al. [23] that the antimicrobial activity of the plant extracts
has a positive correlation with their phenolic and flavonoid contents.

TABLE 1. Phytochemical Screening of Ethanol Extract and fractions Resulted from Fractionation
Results
Compounds Ethanol Hexane Ethyl acetate Butanol Water
Extract fraction fraction fraction fraction
Terpenoids + + - - -
Steroids + + + - -
Alkaloids + - + - -
Flavonoids + - + + +
Phenols + + + + +
Saponins - - - - -
Information: ( + ) : detected, ( - ) : not detectable (no discoloration or precipitation or foam formation)

Antimicrobial Activity Assay

Results of antibacterial activity assay can be seen at Table 2 and Figure 1. At concentration of 20%, the rhizome
ethyl acetate fraction strongly inhibited Escherichia coli, Staphylococcus aureus, and Candida albicans with the
inhibition zones of 19.25 ± 0.20; 14.00 ± 0.17; 14.00 ± 0.20 mm respectively, followed by n-butanol fraction that

020054-4
inhibited the microbes with the inhibition of 15.50 ± 0.22; 12.00 ± 0.15; 12.50 ± 0.19 mm. Next, n-hexane fraction
inhibited the growth of E.coli, S. aureus, and C. albicans with the inhibition of 13.00 ± 0.22; 10.00 ± 0.18; and 9.75
±0.17 mm, while there was no inhibition of water fraction towards all the microbes.Therefore, only n-hexane, ethyl
acetate, and n-butanol fraction was determined their MIC values.

TABLE 2. Antimicrobial Assay of Hexane, Ethyl Acetate, Butanol, and Water F ractions
Against E. coli, S. aureus, dan C. albicans at Concentration of 20%
Average inhibition zone (mm)
No. Fractions
E. coli S. aureus C. albicans
1. n-Hexane 13.00 ±0.22 10.00 ± 0.18 9.75 ± 0.17
2. Ethyl acetate 16.25 ± 0.20 14.00 ±0.17 14.00 ±0.20
3. n-Butanol 15.50 ± 0.22 12.00 ± 0.15 12.50 ± 0.19
4. Water 0 0 0

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FIGURE 1. Antimicrobial assay of fractions (A) E .coli, (B) S. aureus, and (C) C. albicans at concentration 20%. 1) control; 2)
water fraction; 3) n-hexane fraction; 4) n-butanol fraction; 5) ethyl acetate fraction

To determine the MIC of n-hexane, ethyl acetate, and n-butanol fractions, concentrations of 2, 4, 6, 8, 19, and 15
% (b/v) were prepared. The results show that n-hexane fraction at a concentration of 8, 10, and 15 % were able to
inhibit the growth of E. coli with the inhibition zone of 7.5± 0.21, 10.5± 0.14, and 11.75± 0.17 mm respectively after
24 hours of treatment. Meanwhile, there was no inhibition of that towards S. aureus and C. albicans (Table 3).
Therefore, the MIC of n-hexane extract against E-coli was 8 %, while the concentration of 20% was the MIC of the
extract toward S. aureus and C. albicans.

TABLE 3. Antimicrobial Assay of n-Hexane fractions with Various

Concentration Towards E. coli, S. aureus, and C. albicans
Concentration Average inhibition zone (mm)
No
(%) E. coli S. aureus C. albicans
1 15 11.75 ± 0.17a* 0 0
b
2 10 10.5 ±0.14 0 0
3 8 7.5 ± 0.2c 0 0
4 6 0d 0 0
5 4 0 0 0
6 2 0 0 0
* Values followed by the same letters in the same column are not significantly different according to the
Duncan’s Multiple Range Test at P<5%.

020054-5
 The activity of n-hexane fraction was probably caused by D- or E-asarone (essential oil). Balakumbahan et al.
[17] revealed that D- and β–asarones is responsible for its antimicrobial activities. Rita et al. [20] reported that the
essential oil of A. calamus rhizomes strongly inhibited the growth of C. albicans with the MIC of 1 %. However, the
n-hexane fraction in this study was no inhibition toward C. albicans. Besides the essential oils, there were other non-
polar components which were dissolved in n-hexane. The combination of these compounds was likely to reduce,
even negate the antimicrobial activity. This means that the combination of the compounds was antagonistic.
Antimicrobial assay of ethyl acetate fraction with various concentrations against E. coli, S. aureus, and C.
albicans was presented in Table 4, while that of the n-butanol fraction is presented in Table 5. From the Table 4, it
can be seen that the Ethyl acetate fraction inhibited the growth of E. coli with the inhibition of 9.00± 0.15, 10.50±
0.22, 13.00± 0.20, and 16.25± 0.12 at the concentration of 6, 8, 10, and 15 % respectively. At the concentration of 4
%, there was no inhibition. Meanwhile, the fraction has able to inhibit S. aureus and C. albicans at a concentration
from 8%. From the result, it can be revealed that the MIC of ethyl acetate fraction against E. coli, S. aureus, and C.
albicans was successively 6, 8, 8 %. Table 4 shows that the inhibition increased with the increase of concentration
fractions, the inhibitions were significantly different with the different concentration applied.
From Table 5, it can be seen that the minimum concentration of n-butanol fraction towards E. coli, S. aureus, and
C. albicans was 6, 10, 8 % respectively. However, at the same concentration, the inhibition of the ethyl acetate
fraction was higher than the n-butanol fraction. Susanah et al. [13] reported that ethanol extract of A. calamus was
strongly inhibited the growth of E. coli, S. aureus, and C. albicans at a concentration of 10% with an inhibition zone
of 16.60, 13.60, and 15.27 mm respectively. It shows that the antibacterial activity of ethanol extract was stronger
than that of all fractions. This shows that the compounds from ethanol were synergistic.

TABLE 4. Antimicrobial Assay of Ethyl Acetate Fraction with Various

Concentration Towards E. coli, S. aureus, and C. albicans
Concentration Average inhibition zone (mm)
No E. coli S. aureus C. albicans

07 September 2023 11:30:32

(%)
1 15 16.25 ± 0.12a* 12.50 ± 0.15a* 12.25 ± 0.20a*
2 10 13.00 ± 0.20b 11.25 ± 0.10b 11.25 ± 0.05b
3 8 10.50 ± 0.22c 9.30 ± 0.15c 8.50 ± 0.12c
4 6 9.00 ± 0.15d 0d 0d
5 4 0e 0 0
6 2 0 0 0
* Values followed by the same letters in the same column are not significantly different according to the
Duncan’s Multiple Range Test at P<5%.

TABLE 5. Antimicrobial Assay of the n-Butanol fraction with Various

Concentration Towards E. coli, S. aureus, and C. albicans
Concentration Average inhibition zone (mm)
No
(%) E. coli S. aureus C. albicans
1 15 12.50 ± 0.15 a* 9.20 ±0.17a* 12.00 ± 0.17a*
2 10 11.50 ± 0.11b 8.70 ± 0.04b 11.00 ± 0.15b
c c
3 8 10.50 ± 0.12 0 8.00 ± 0.18c
d
4 6 8.70 ± 0.19 0 0d
e
5 4 0 0 0
6 2 0 0 0
* Values followed by the same letters in the same column are not significantly different according to the
Duncan’s Multiple Range Test at P<5%.

Determination of Total Phenolic and Flavonoid Contents

The curves of the calibration of quercetin to determine total flavonoid contents are presented in Figures 2. It
obtained the calibration equation of y = 0.0313x + 0.0272 (R 2= 0.9961), while that of gallic acid to determine total
phenolic contents can be seen in Figure 3. It obtained the calibration equation of y = 0.051x + 0.014 (R2=0.9980).
The calculation to determine the total flavonoid and phenolic contents are summarized in Table 6. The total

020054-6
flavonoid contents of n-hexane, ethyl acetate, n-butanol, and water fractions were 142.23, 2978.34, 399.07, and
14.80 mg QE/100g fractions successively. The total phenolic contents were 237.81, 1820.51, 329.71, 74.36 mg
GAE/100 g fractions.

A 0.700
0.646
b 0.600
s 0.500 0.527
o
0.400
r y = 0.031x + 0.027
b 0.300 0.282 R² = 0.9961
a 0.200
0.169
n 0.100 0.102
c
0.000 0.000
e
0.0 5.0 10.0 15.0 20.0 25.0
Concentration of Quercetin (mg/L)

FIGURE 2. Calibration Curve of Standard Quercetin

07 September 2023 11:30:32

1.200
A
b 1.000 1.027
s 0.848
0.800
o
0.600 0.628
r
b 0.400 0.440 y = 0.0514x + 0.0146
a R² = 0.9989
0.200 0.232
n 0.112
c 0.000 0.000
e 0.0 5.0 10.0 15.0 20.0 25.0
Concentration of Gallic acid mg/mL

FIGURE 3. Calibration Curve of Standard Gallic Acid

The contents of flavonoid and phenolic compounds in ethyl acetate fraction were the highest, followed by n-
butanol, n-hexane, and water fractions respectively. This phenomenon was the same as antibacterial results.
Therefore, the result confirmed the state of Mahboubi et al. [23] that the antimicrobial activity of the plant extracts
has a positive correlation with their phenolic and flavonoid contents. Hendra et al. [25] revealed that the mechanism
of flavonoids as antimicrobials can be divided into 3 which inhibits nucleic acid synthesis, inhibits membrane
function cells, and inhibit energy metabolism. A and B rings of flavonoids play an important role in the process of
hydrogen interconnection or bonding by accumulating nucleic acid bases which inhibit the formation of DNA and
RNA. The hydroxyl position at position 2 ', 4' or 2 ', 6' hydroxylated at ring B and 5.7 hydroxylated at ring A plays

020054-7
an important role in the antibacterial activity of flavonoids. The mechanism of flavonoids inhibits cell membrane
function by disrupting the permeability of cell membranes and inhibiting enzyme bonds such as ATPase and
phospholipase. Flavonoids inhibit the cytochrome C reductase, therefore the formation of metabolism is inhibited
[26].
The antimicrobial mechanism of phenolic compounds is by denaturing cell proteins. The hydrogen bond formed
between phenol and protein causes the protein structure to be damaged. The hydrogen bond will affect the
permeability of the cell wall and cytoplasmic membrane because both are composed of proteins. Permeability of cell
walls and cytoplasmic membranes that are disrupted can cause an imbalance of macromolecules and ions in cells so
that cells become lysis [27].

TABLE 6. Determination of Total of Flavonoid and Phenolic Contents

Sample Flavonoid Contents
Sample
Weight Abs (Y) Kons (x)
Fractions concentration Dilution
(mg) mg/L % mg/100 g
mg/5 mL

Total Flavonoid Contents(y = 0.0313x + 0.0272)

Hexane 53.80 10.76 0.123 3.0607 5 0.1422 142.23
Ethyl acetate 50.90 10.18 0.217 6.0639 50 2.9783 2978.34
n-Butanol 55.40 11.08 0.304 8.8435 5 0.3991 399.07
Water 51.60 10.32 0.075 1.5272 1 0.0148 14.80

Total Phenolic Contents (y = 0.051x + 0.014)

07 September 2023 11:30:32

Hexane 121.80 24.36 0.759 14.4825 4 0.2378 237.81
Ethyl acetate 52.30 10.46 0.985 19.0431 10 1.8206 1820.57
n-Butanol 103.90 20.78 0.895 17.1284 4 0.3297 329.71
Water 288.00 57.6 0.565 10.7082 4 0.0744 74.36

CONCLUSION
This study revealed that the A. calamus rhizome ethyl acetate fraction has the highest antibacterial activity
against Escherichia coli, Staphylococcus aureus, and Candida albicans, followed by n-butanol and n-hexane
fractions respectively, while there is no inhibition of water fraction towards the microbes. The antibacterial activity
has a positive correlation with the total content of flavonoids and phenolics. A. calamus rhizome contains high
antimicrobial property and can be further be explored for the isolation of its bioactive compounds.

ACKNOWLEDGMENT
This work was supported by a grant from Udayana University. We wish to express our gratitude to head of
Research and Community Institutions of Udayana University facilitating all the needs in the disbursement of
research funds.

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