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Citrus x microcarpa bunge fruit extract as antibacterial against staphylococcus aureus

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O Roanisca
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Robby Gus Mahardika at University of Bangka Belitung
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Abstract and Figures

Citrus x microcarpa Bunge is a plant that is used optimally by the people of Bangka Belitung known as the ‘Jeruk Kunci’. Utilization of this plant in the fruit is used as an acid enhancer in food and is made in the form of syrup. So that the jeruk kunci fruit waste is widely available and has not been utilized.based on literature, Citrus x microcarpa contains flavonoids and a monoterpeneshydrocarbon. Bioactivity which belongs asantimicrobial, antibacterial and antioxidant. Therefore, jeruk kunci fruit waste has the potential to be developed as an antibacterial herbal medicine based on literature studies. Citrus x microcarpa Bunge extract was obtained by maceration for 3 x 24 hours using ethanol solvent. Then phytochemical testing was conducted qualitatively using several reagents and quantitative phytochemical testing was carried out using functional group analysis contained in the extract uses the Fourier Transform Infrared (FT-IR) at the wavenumber region 4000-400 cm-1. Then, determination of antibacterial activity using the disk diffusion method. Based on FT-IR spectrum data analysis Citrus x microcarpa extract contains tannins. The results of antibacterial testing of Citrus x microcarpa Bunge extract obtained concentration of 20%, 40% and 60% has a relatively strong antibacterial inhibition. whereas for 80% and 100% concentrations the ability to inhibit is very strong.
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Citrus x microcarpa
bunge fruit extract as antibacterial against
staphylococcus aureus
To cite this article: O Roanisca and R G Mahardika 2020 IOP Conf. Ser.: Earth Environ. Sci. 599 012043
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2nd International Conference on Green Energy and Environment (ICoGEE 2020)
IOP Conf. Series: Earth and Environmental Science 599 (2020) 012043
IOP Publishing
doi:10.1088/1755-1315/599/1/012043
1
Citrus x microcarpa bunge fruit extract as antibacterial
against staphylococcus aureus
O Roanisca* and R G Mahardika
Department of Chemistry, Faculty of Engineering, Universitas Bangka Belitung,
Kampus Terpadu UBB Balunijuk, Merawang, Bangka Regency, Bangka Belitung
Province, 33172, Indonesia
*E-mail: occaroanisca@gmail.com
Abstract. Citrus x microcarpa Bunge is a plant that is used optimally by the people of Bangka
Belitung known as the 'Jeruk Kunci'. Utilization of this plant in the fruit is used as an acid
enhancer in food and is made in the form of syrup. So that the jeruk kunci fruit waste is widely
available and has not been utilized.based on literature, Citrus x microcarpa contains flavonoids
and a monoterpeneshydrocarbon. Bioactivity which belongs asantimicrobial, antibacterial and
antioxidant. Therefore, jeruk kunci fruit waste has the potential to be developed as an
antibacterial herbal medicine based on literature studies. Citrus x microcarpa Bunge extract
was obtained by maceration for 3 x 24 hours using ethanol solvent. Then phytochemical testing
was conducted qualitatively using several reagents and quantitative phytochemical testing was
carried out using functional group analysis contained in the extract uses the Fourier Transform
Infrared (FT-IR) at the wavenumber region 4000-400 cm-1. Then, determination of
antibacterial activity using the disk diffusion method. Based on FT-IR spectrum data analysis
Citrus x microcarpa extract contains tannins. The results of antibacterial testing of Citrus x
microcarpa Bunge extract obtained concentration of 20%, 40% and 60% has a relatively strong
antibacterial inhibition. whereas for 80% and 100% concentrations the ability to inhibit is very
strong.
1. Introduction
Infectious diseases caused by bacteria are still a problem both in developing countries and in
developed countries. Staphylococcus aureus is one of the most common bacteria that cause infections
in the world. This bacterium can live on human skin and is very easy to breed at an optimum
temperature of 30oC. Diseases caused by S. aureus include respiratory infections, abscesses, skin
inflammation, mastitis, impetigo, and food poisoning that cause diarrhea accompanied by nausea and
vomiting [1],[2].
Antibacterial is a compound that can inhibit bacterial growth. Antibacterial can be a synthetic
compound and natural compounds. Antibacterial that can be used to inhibit the growth of
Staphylococcus aureus such as erythromycin, cloxacillin and dicloxacillin. The use of synthetic
antibiotics allows for resistance. Natural compounds found in organisms are known as secondary
metabolites. Secondary metabolites that act as antibacterial include alkaloids, phenols and flavonoids.
The mechanism of action of secondary metabolites, among others, by damaging bacterial cell walls,
2nd International Conference on Green Energy and Environment (ICoGEE 2020)
IOP Conf. Series: Earth and Environmental Science 599 (2020) 012043
IOP Publishing
doi:10.1088/1755-1315/599/1/012043
2
changing membrane permeability, inhibiting the work of enzymes, and disrupt the process of protein
synthesis [3].
Citrus x microcarpa Bunge is widespread in the Bangka Belitung area and is known by the name of
the jeruk kunci. Jeruk kunci are used as an acid enhancer in foods, and are processed into key orange
syrup, so that the jeruk kunci fruit waste is abundant and has not been utilized. Research on
phytochemicals and bioactivity of Citrus x microcarpa Bunge found that C. x microcarpa Bunge
contains flavonoids, alkaloids, polyphenols, monoterpenes hydrocarbon such as limonene, sabinen,
citronellal, linalool and hedycaryol with bioactivity as antimicrobial, antibacterial, antioxidant and
antifeedant [4-8]. Flavonoid compounds also act as antibacteria. Therefore, this research will examine
the antibacterial bioactivity of jeruk kunci fruit waste against Staphylococcus aureus
2. Materials and methods
2.1. Material and equipment
The materials used in this study were jeruk kunci fruit waste from industrial waste of jeruk kunci
syrup Air Duren Village, dimethyl sulfoxide (DMSO), nutrient agar, nutrient broth, ethanol, paper
discs, amoxicillin, Mueller-Hinton agar (MHA), aquades, alumunum foil, cotton, and label paper.
Equipment used includes petri dishes, test tubes, erlenmeyers, micro pipettes, rotary evaporators,
vacuum pumps, Buchner funnel, vial bottle volumetric flask and spatula.
2.2. Sample Preparation
Waste of fresh jeruk kunci fruits as much as 1 kg is cleaned by washing with running water and then
dried in direct sunlight. A 450 gram dry sample was blended using a blender, to increase contact
between the sample and the solvent [9].
2.3. Extraction
450 gram sample powder was macerated for 3 x 24 hours with comparison of sample with solvent
1:10. Every 1 x 24 hours, filtering and solvent replacement are carried out so that the extraction
process runs optimally [10]. The filtrate obtained was evaporated using a rotary evaporator, so that a
thick extract was obtained [11]. The next step, the viscous extract obtained was tested for antibacterial
against Staphylococcus aureus and confirmed the presence of secondary metabolite functional groups
using Fourier Transform Infrared (FT-IR) in the wave number range 4000-400 cm-1.
2.4. Antibacterial testing
Antibacterial testing is carried out using the agar diffusion method. A bacterial suspension is applied
to the petri dish which contains a solid nutrient agar. Next 6 mm disc paper saturated with 20%, 40%,
60%, 80%, and 100% concentration test solution is placed on a petri dish. The same work for disc
paper as a positive control and negative control. Positive control in this test uses amoxillin and
negative control uses DMSO solvent. Then the petri dish was incubated for 24 hours at 370C.
Measurement of antibacterial activity is done by measuring clear zones formed using calipers.
Furthermore, the clear zone diameters formed were classified into four categories, namely <5 mm
weak, 5-10 mm moderate, 10-20 mm strong and> 20 mm very strong [12].
3. Result and Discussion
Citrus x microcarpa Bunge is widespread in the Bangka Belitung area and is known by the name of
the jeruk kunci (Figure 1). Based on research that has been done. Citrus x microcarpa contains tannin
compounds based on tests with FeCL3 with a greenish-black color change.
2nd International Conference on Green Energy and Environment (ICoGEE 2020)
IOP Conf. Series: Earth and Environmental Science 599 (2020) 012043
IOP Publishing
doi:10.1088/1755-1315/599/1/012043
3
Figure 1. Fruit of Citrus x microcarpa Bunge
Spectrum FTIR of Citrus x microcarpa on Figure 2. FTIR functions to find out the functional
groups contained in the synthesized product.
Figure 2. FTIR Spectrum of Citrus x microcarpa Bunge
Widened absorption from 3400 to 3100 cm-1 indicates the vibration of the -OH (hydroxyl) group,
the widening band caused by the interaction of intermolecular hydrogen bonds. This hydroxyl group
can also originate from the phenol group (Ar-OH) which is strengthened by absorption at the 738 cm-1
wave number. The uptake of this OH OH group may also overlap with C-H aromatic stretching uptake
which is usually at 3159-3050 cm-1. This indicates the presence of aromatic groups in the extract. The
presence of aromatics is also supported by the presence of vibration C = C stretching at 1614 cm-1.
The analysis showed that Citrus x microcarpa Bunge extract contained aromatic compounds in the
form of phenolic compounds or polyphenols such as tannins.
Bands with wave numbers 2923 cm-1 indicate the presence of CH3 (methyl) groups and CH as
asymmetric vibrations (methylene). The existence of this group is also supported by the absorption of
wave numbers from 1448 to 1334 cm-1. This indicates that the Citrus x microcarpaextract ¬ contains
methyl, methylene and metin groups.
Based on FT-IR spectrum data analysis (Figure 2. and Table 2), Citrus x microcarpaextract
contains compounds that have Ar-OH (phenolic), -OH (hydroxyl), and C = C (aromatic) groups. This
indicates that the functional group is a functional group on the structure of polyphenols such as
tannins.
2nd International Conference on Green Energy and Environment (ICoGEE 2020)
IOP Conf. Series: Earth and Environmental Science 599 (2020) 012043
IOP Publishing
doi:10.1088/1755-1315/599/1/012043
4
Table 1. Vibrational mode of C. x microcarpaExtract
Wavenumber (cm-1)
Vibration modes
C. x microcarpa
3351
-OH stretch alcohol/phenol
2923
C-H stretch CH3/asymmetric CH2
1614
C=C aromatic
1448
CH2 bend
1334
C-H bend
1210
C-C stretch
1031
C-O alkohol stretch
738
C-H aromatic bend
Antibacterial testing of C. x microcarpa Bunge extract (jeruk kunci) was carried out using the disc
diffusion method. The potential for antibacterial activity is known by measuring in the clear zone area
around the disc paper. After testing antibacterial against gram-positive Staphylococcus aureus with
test material in the form of ethanol extract of jeruk kunci fruits waste, DMSO (negative control) and
amoxillin (positive control) solvents, the data shown in the table below.
Table 2. Inhibition zone of ethanol extract of Citrus x microcarpa Bunge on the growth of
Staphylococcus aureus
Based on the above data, ethanol extract of jeruk kunci fruit waste has the potential to inhibit the
growth of Staphylococcus aureus. Positive control in this study using amoxillin with inhibition zone
diameter of 18.45 mm and negative control using DMSO solvent did not show any inhibition of the
growth of Staphylococcus aureus bacteria.
The antibacterial potential of ethanol extract of jeruk kunci fruits waste in inhibiting the growth of
S. aureus began to be seen at the lowest concentration of 20% of 12, 36 mm. At extract concentration
of 40% has a inhibitory power of 17.37 mm. The extract concentration of 60% formed a clear zone of
19.61 mm, and at the extract concentration of 80% and 100% formed a clear zone of 22.90 mm and
26.63 mm respectively. The growth of S. aureus decreased with increasing concentration of jeruk
kunci fruits waste extracts. Inhibition of the growth of S.aureus over positive control was shown at
extract concentrations of 60%, 80% and 100%. Based on these data extract concentrations of 20%,
40% and 60% have a relatively strong bacterial inhibition strength, at a concentration of 80% and
100% the inhibitory strength of bacteria is very strong. Research on pure compounds 3', 4', 5,6,7,8-
hexamethoxyflavone isolated from Citrus microcarpa from Manila did not show any antibacterial
activity against S. aureus [4].
Based on FTIR spectrum data analysis, Citrus x microcarpa Bunge contains tannins. The tannin /
phenol hydroquinone compound as an antibacterial by inhibiting the transcriptase enzyme and the
DNA topoisomerase being not formed. The mechanism of action of other tannins as antibacterials by
activating cell adhesin, activating enzymes and disrupting protein transport. Disruption in the protein
synthesis of bacterial cells due to the presence of tannin compounds will be fatal, leading to bacterial
cell death [13].
4. Conclusion
Citrus x microcarpa Bunge extract contains compounds that have Ar-OH (phenolic), -OH (hydroxyl),
and C = C (aromatic) groups. This indicates that the functional group is a functional group on the
Concentration of extract
Average Inhibition Zone Diameter (mm)
20%
12.36
40%
17.37
60%
19,.61
80%
22.90
100%
26.63
2nd International Conference on Green Energy and Environment (ICoGEE 2020)
IOP Conf. Series: Earth and Environmental Science 599 (2020) 012043
IOP Publishing
doi:10.1088/1755-1315/599/1/012043
5
structure of polyphenols such as tannins. Tannins including phenolic groups like flavonoid. The results
of antibacterial testing of Citrus x microcarpa Bunge extract obtained concentration of 20%, 40% and
60% has a relatively strong antibacterial inhibition. whereas for 80% and 100% concentrations the
ability to inhibit is very strong.
References
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[3] Mickymaray S 2019 Efficacy and Mechanism of Traditional Medicinal Plants and Bioactive
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[13] Othman L Sleiman A and Abdel-Massih R M 2019 Antimicrobial Activity of Polyphenols and
Alkaloids in Middle Eastern Plants Frontiers Microbial 10 pp 1-28.
Acknowledgments
We gratefully acknowledge the funding from Universitas Bangka Belitung through the RKAKL FT
for the publication of this paper.
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  • Dec 2019
Traditional medicinal plants have been cultivated to treat various human illnesses and avert numerous infectious diseases. They display an extensive range of beneficial pharmacological and health effects for humans. These plants generally synthesize a diverse range of bioactive compounds which have been established to be potent antimicrobial agents against a wide range of pathogenic organisms. Various research studies have demonstrated the antimicrobial activity of traditional plants scientifically or experimentally measured with reports on pathogenic microorganisms resistant to antimicrobials. The antimicrobial activity of medicinal plants or their bioactive compounds arising from several functional activities may be capable of inhibiting virulence factors as well as targeting microbial cells. Some bioactive compounds derived from traditional plants manifest the ability to reverse antibiotic resistance and improve synergetic action with current antibiotic agents. Therefore, the advancement of bioactive-based pharmacological agents can be an auspicious method for treating antibiotic-resistant infections. This review considers the functional and molecular roles of medicinal plants and their bioactive compounds, focusing typically on their antimicrobial activities against clinically important pathogens.
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Antibacterial Effectiveness Of Formulations Nanoemulsion Cratoxylum Glaucum Korth. Extract
Article
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  • Oct 2019
Cratoxylum glaucum Korth. is a members of the family Hypericaceae which are spread in Indonesia, one of which is found in the forest of the Bangka Belitung Islands. Cratoxylum glaucum Korth is often used by the people of Bangka Belitung as a flavoring ingredient from ancient times until now it is still consumed and utilized by people around Bangka Belitung. One source of natural antioxidants is the top of the crust. The effort of increasing its activity is very strong, so in this study we try to develop extraction of Cratoxylum glaucum made in the form of nanoemulsion. This study aims to determine the comparison of Cratoxylum glaucum extract in nanoemulsion preparations with extracts as antibacterial. Nanoemulsion is made with the composition of Virgin Coconut Oil (VCO) as an oil phase, tween 80 as surfactant and aquades as a water phase with a ratio of 2.5: 10: 25. The nanoemulsion extract of Cratoxylum glaucum have a size 422.9 nm and PI value 0.493. The antibacterial activity of nanoemulsion formulation has inhibitory growth power in moderate categorized Staphylococcus aureus bacteria, whereas in the extract of Cratoxylum glaucum it has the inhibitory growth of active Staphylococcus aureus bacteria.
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Antimicrobial Activity of Polyphenols and Alkaloids in Middle Eastern Plants
Article
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  • May 2019
Antibiotic-resistant microorganisms have been an ever-growing concern over the past years. This has led researchers to direct their attention onto plants to be able to discover new possible antimicrobial compounds. The Middle East encompasses a wide spectrum of plant diversity with over 20,000 different species in habitats ranging from deserts to snow-capped mountains. Several plant secondary metabolites and their derivatives have been identified as possible antimicrobial agents. Among the secondary metabolites studied, alkaloids and polyphenols have shown strong antimicrobial activity. Polyphenols are one of the most numerous and diverse group of secondary metabolites; their antioxidant properties provide the basis for antimicrobial effects. Alkaloids provided the underlying structure for the development of several antibiotics with a diverse range of action. The ability of some plant secondary metabolites to act as resistance-modifying agents is a promising field in mitigating the spread of bacterial resistance.
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Traditional Small-Size Citrus from Taiwan: Essential Oils, Bioactive Compounds and Antioxidant Capacity
Article
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  • May 2017
Background: The calamondin (Citrus microcarpa Bunge) and the kumquat (Fortunella crassifolia Swingle) are two small-size citrus fruits that have traditionally been consumed in Taiwan; however, there has been a lack of scientific research regarding the active compounds and functionalities of these fruits. Methods: Analysis of volatile composition of essential oil and phytosterol was carried out using Gas Chromatography–Mass Spectrometry (GC-MS). Flavonoid and limonoid were analyzed by High Performance Liquid Chromatography (HPLC). Moreover, antioxidant capacity from their essential oils and extracts were assessed in vitro. Results: The compositions of the essential oils of both fruits were identified, with the results showing that the calamondin and kumquat contain identified 43 and 44 volatile compounds, respectively. In addition, oxygenated compounds of volatiles accounted for 4.25% and 2.04%, respectively, consistent with the fact that oxygenated compounds are generally found in high content in citrus fruits. In terms of flavonoids, the calamondin exhibited higher content than the kumquat, with disomin-based flavonoids being predominant; on the other hand, phytosterol content of kumquat was higher than that of calamondin, with amyrin being the dominant phytosterol. Both of them contain high amounts of limonoids. The ethanol extracts and essential oils of small-sized citrus fruits have been shown to have antioxidant effects, with those effects being closely related to the flavonoid content of the fruit in question. Conclusions: The present study also reviewed antioxidant activity in terms of specific bioactive compounds in order to find the underlying biological activity of both fruits. The calamondin and kumquat have antioxidant effects, which are in turn very important for the prevention of chronic diseases.
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Essential Oils from the Malaysian Citrus (Rutaceae) Medicinal Plants
Article
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  • Jun 2016
This review article appraises the extraction methods, compositions, and bioactivities of the essential oils from the Citrus species (family: Rutaceae) endemic to Malaysia including C. aurantifolia, C. grandis, C. hystrix, and C. microcarpa. Generally, the fresh peels and leaves of the Citrus species were extracted using different methods such as steam and water distillation, Likens-Nikerson extraction, solvent extraction, and headspace solid-phase micro-extraction (HS-SPME). Most of the Citrus oils were found to be rich in monoterpene hydrocarbons with limonene (1) as the major component identified in the peels of C. aurantifolia (39.3%), C. grandis (81.6%–96.9%), and C. microcarpa (94.0%), while sabinene (19) was the major component in the peels of C. hystrix (36.4%–48.5%). In addition, citronellal (20) (61.7%–72.5%), linalool (18) (56.5%), and hedycaryol (23) (19.0%) were identified as the major components in the oil of C. hystrix leaves, C. grandis blossom and C. microcarpa leaves, respectively. The C. hystrix essential oil has been experimentally shown to have antimicrobial and antifeedant activities, while no bioactivity study has been reported on the essential oils of other Malaysian Citrus species.
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Antimicrobial flavonoid from Citrus microcarpa
Article
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  • Jan 2006
Air-dried leaves of Citrus microcarpa Bunge afforded 3',4',5,6,7,8-hexamethoxyflavone (1) and squalene. The structure of 1 was elucidated by extensive 1D and 2D NMR spectroscopy. Antimicrobial tests on 1 indicated that it has moderate activity against the fungus, C. albicans and low activity against the bacteria, P. aeruginosa and B. subtilis and fungi, T. mentagrophytes and A. niger.
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Microwave-Assisted Extraction of Polyphenol Content from Leaves of Tristaniopsis merguensis Griff.
Article
  • Jan 2020
Tristaniopsis merguensis Griff. is a species of the Myrtaceae family and has been widely used by people of Bangka Belitung as a traditional medicine to reduce cholesterol, gastric pains, and improve cardiac performance. Extraction methods are the crucial efficacy of herbal medicine. The conventional method, like maceration, takes a long time. In this study, the leaves of Tristaniopsis merguensis were extracted using Microwave-Assisted Extraction (MAE) to reduce extraction time. The extraction using MARS (Microwave Accelerated Reaction System) 6 by CEM Corporation with time variation times of 5, 10, 15, 30 min with temperature of 60, 80, 100oC at 1200 W. The yield using acetone extraction of Tristaniopsis merguensis leaves increases with time and temperature. The extraction dependent on solvent extraction, polar solvent like ethanol, and methanol were higher than semi-polar solvents like acetone and ethyl acetate. The polyphenol content of acetone extract using MAE (10 min, 80oC) was found to be 234.67 mg Gallic Acid Equivalent per gram (GAE/g); it was higher than acetone extract using maceration. The phytochemical results show there are no difference in the active compound using MAE and maceration, i.e. alkaloids, tannin, and flavonoids. Yield extraction, time, and phytochemical results of MAE are more favorable than a maceration.
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