INTRODUCTION:

Infection due to bacteria can inhibit wound healing process on the skin of patients who are undergoing treatment in the hospital, such as patients with surgery, urinary catheters, and long-term infusions. Some of the bacteria that cause nosocomial infections of the skin include Staphylococcus aureus, Streptococcus pyogenes, Acinetobacter sp., Pseudomonas aeruginosa. Among the bacteria that cause skin infections, Staphylococcus aureus is one that is often found in skin infections.¹ Many infectious bacteria are resistant to one or several antibiotics².

Inflamed skin is characterized by an itchy skin rash. Colonization of Staphylococcus aureus on the skin can be found in more than 90% of skin with atopic dermatitis due to impaired skin barrier function and natural immunity. Meanwhile, in healthy individuals there are only 5-25% of the population. Staphylococcus aureus bacteria may cause atopic dermatitis and skin inflammation³ by secreting several toxins that act as superantigens and activate macrophages and T cells.⁴

Staphylococcus aureus bacteria continue to grow and cases of antibiotic resistance have been reported to have increased since the use of penicillin in 1942.^5,6 Penicillin antibiotics act by inhibiting protein binding penicillin (PBP) which plays an important role in the synthesis of bacterial cell walls. By inhibiting PBP the bacteria will die due to osmosis.⁶ The bacteria begin to produce the enzyme penicillinase, with a specific type, the beta-lactamase. This enzyme hydrolyzes the antibiotic and makes the antibiotic ineffective. Pharmaceutical companies produce penicillinase-resistant semi-synthetic antibiotics, known as methicillin. The mechanism of action of methicillin is similar to penicillin.⁷ However, in a short time, Staphylococcus aureus will show resistance to methicillin treatment. This strain first appeared in the United Kingdom and is known as Methicillin-resistance Staphylococcus aureus.^6,8

Until now, many antibiotics used to treat wound infections caused by pathogenic bacteria have become resistant. The clinical efficacy of many existing antibiotics is being threatened by the emergence of multidrug-resistant pathogens.⁹ Cases of increased antibiotic resistance to microorganisms have been reported.¹⁰ In accumulation to this predicament, antibiotics are occasionally coupled with adverse effects on the host together with immune suppression, hypersensitivity as well as allergic reactions.¹¹ One way to prevent antibiotics resistance of pathogenic bacteria is by using new compounds that are not based on existing synthetic antimicrobial agents.¹² Until now, many researchers have concentrated on developing research to find antimicrobial ingredients from herbal plants. Apart from being considered safer because of its low toxicity, this measure is also an attempt to overcome the increase in antibiotic resistance.¹³ Traditional medicine is revealed by an extensive activity of research on different plant species and their therapeutic principle in the world.¹⁴

Traditional medicine is still the mainstay, mainly in developing countries. Traditional medicine of better cultural acceptability better compatibility eith the human body and fewer side effects. However, the last few years have seen a major increase in their use in the developed world.⁹ Numerous investigation have been carried out in invidual plants and the respective antimicrobial agents have been identified in a gratifying number of case. From various studies, it is clear that the chemical structures of these agents belong to the most commonly encountered classes of higher plant secondary matabolites.¹⁵

Pure compounds or herbal plant extracts containing various chemical substances can be used as important indications as natural antimicrobials.¹³ Besides being safer, the material is easily available and is expected to have a positive impact on human health.¹⁶ The compounds that show the most activity are phenolics and flavonoids.^{14,
17, 18} Part of plants that van be used as medicine are leaves, seeds, stems, roots, fruit.^17,19 The part of these plants can be extracted, boiled, powdered to take the active compound.^17,20 The diversity of secondary metabolites produced by plants due to interactions between plants and environment.¹⁸

Lime peel is a part that is rarely used and considered as waste.²¹ Lime peel is divided into epicarp or flavedo and mesocarp or albedo. Flavedo is the outer part of the lime peel which is yellowish green in color, while albedo is the soft and white inner layer of the lime peel. Lime peels contain high amounts of phenolic compounds, including several flavonoids. Lime peel extract and essential oil are known to exhibit various biological activities, such as antimicrobial and antioxidant activity.²² Thus there is a critical need to develop new drugs with novel mechanisms of action.²³

Lime contains flavonoid compounds with components similar to lemons, ie. hesperidin, eriocitrin, and diosmin. In lime, the most abundant components are flavanones and flavones. In addition, there is the presence of other flavanones (narirutin and neoeriocitrin), as well as flavanones and flavone aglycones (taxifolin and luteolin, respectively). New compounds in lime have also been found, albeit in low levels, namely polymethoxyflavones (natsudaidain, heptamethoxyflavone, nobiletin, sinensetin and tangeretin).²⁴

This study aims to examine the antibacterial activity of MeOH: DMSO (1:1, v/v) extract on the peel of lime (Citrus aurantifolia (Christm.) Swingle) against Methicillin-Resistant Staphylococcus aureus (MRSA).

MATERIAL AND METHODS:

This study was an experimental laboratory study. Data were collected from April 2019 to November 2019. Data analysis regarding the prediction of potential compounds in lime peel was carried out in silico using open access softwares PubChem, PASS SERVER, and STITCH. In vitro data analysis was performed using SPSS version 17.

Lime plant determination:

Lime fruits used in this study were taken from lime plantations in Bumi Aji Village, Batu City, East Java, Indonesia. Plant samples were determined by the Biology Service Unit, Faculty of Science and Technology, Airlangga University, Surabaya, Indonesia. The determination was carried out to reduce the possibility of sample identity errors. If the results of the determination showed that the samples tested were true Citrus aurantifolia species, then the lime fruits were collected.

Lime fruit collection:

From the defined lime plants, the fruits were collected. Ripe lime fruits free from insect infection and other types of damage were collected from lime plantations in Bumi Aji Village, Batu City, East Java, Indonesia.

Lime peel preparation:

The lime fruits were washed several times with clean water. The skin was separated from the pulp, then the lime peel was cut into small pieces using a stainless steel knife, and dried in the shade.

Extraction of lime peels uses multilevel solution polarity:

A total of 2.5kg of lime peel samples were immersed in 6 L of n-hexan solution in a glass container and left for 48 hours, stirred occasionally, then filtered so that the residue and filtrate were obtained. The filtrate was evaporated and a thick extract of n-hexan (non polar) was produced. The residue was soaked again in 6 L ethyl acetate for 48 hours, then filtered and the residue and filtrate were produced. The filtrate was evaporated and a thick ethyl acetate extract (semi polar) was produced. The residue was soaked again with 6 L of methanol (MeOH) for 48 hours and filtered to produce residue and filtrate. The filtrate was evaporated and a viscous (polar) methanol extract was produced. The residue was soaked again with a mixture of methanol and DMSO in a ratio of 1:1 for 48 hours, then filtered and the residue and filtrate were produced. The filtrate was evaporated and a MeOH:DMSO (highly polar) extract was obtained.²⁵ MeOH:DMSO (1:1,v/v) extract was weighed and freeze-drying process was carried out.

Flavonoid screening test in extract:

A total 2g of MeOH:DMSO (1:1, v/v) extract of lime peel was put into a test tube and added 3 drops of concentrated HCl and 2mg of magnesium powder. The presence of flavonoids is indicated by the color red, yellow, or orange depending on the structure of flavonoids contained inte sampel.²⁶

Antibacterial potential prediction of 12 flavonoid compounds in extract:

Twelve active of flavonoid compounds in MeOH:DMSO (1:1, v/v) extract of lime peel were adapted from the results of research by Nogata et al (2006). The search for compounds to be analyzed for their potential was carried out by means of PubChem database to obtain compound structures in SMILE data format. The compound data in SMILE format were used for further analysis. The potential antibacterial for compounds in extract was predicted based on the similarity of functional groups of other compounds that have known potentials using PASS SERVER.

In vitro antibacterial activity of MeOH:DMSO (1:1, v/v) extract of lime peel against MRSA:

The agar well diffusion test was carried out to determine the antibacterial activity of the lime peel extract. In the first step, bacterial suspensions were made by taking MRSA bacterial colonies from NAS medium and suspended in sterile 0.85% NaCl. The turbidity of the tested bacterial suspension should have been equivalent to 1.5 x 10⁸ cfu/ml. The second step was to make varied concentrations of MeOH:DMSO (1:1, v/v) extract of the lime peel, ie. 6.25, 12.5, 25, 50, 100, 200, 400 and 800 ppm. The positive control used Penicillin-G 10 U²⁷ and the negative control used MDSO.²⁸ In the third step, as much as 5ml of MHA was poured into a sterile petri dish and allowed to solidify (as the base layer). After solidifying, 3 steel reservoirs were placed and the distance between the reservoirs were adjusted 1.5 - 2cm. Then, the tested bacterial suspension was mixed into 20 ml of MHA media in a test tube. The mixture was poured into a petri dish that had been fitted with steel reservoirs as a second layer (seed layer) and allowed to solidify. The steel reservoirs were lifted aseptically from the petri dish, so that the wells were formed. Each concentration variation of the MeOH:DMSO (1:1, v/v) extract of lime peel and control as much as 100 µl were put into the labeled wells. Petri dishes were incubated at 37^oC for 24 hours in an upright position and the diameter of the inhibition zone formed around the wells was measured.²⁹

Statistic analysis:

The experiment was carried out in vitro. Each treatment was repeated 3 times. Data obtained were statistically tested using one-way ANOVA.

RESULTS:

The results of the determination showed that the lime plant used in this study was certainly a type of Citrus aurantifolia (Chritm.) Swingle from the Rutaceae family. Based on the results of flavonoid screening test, the MeOH:DMSO (1:1, v/v) extract of lime peel peel (Citrus aurantifolia (Christm.) Swingle) contained flavonoid compounds.

Prediction of the potential of flavonoid compound in MeOH:DMSO (1:1, v/v) extract of lime peel were adapted from the results of research by Nogata et al (2006) carried out through PASS SERVER showed as many as eight compounds based on prediction in computation that have antibacterial activity with a Probability activity value ≥0.6 and the other four compounds have antibacterial activity with a Probability activity ≤0.6. The results of computational prediction of the extract's antibacterial potential and penicillin are presented in Table 1.

Table 1: Potential of flavonoid compounds as antibacterial

Compounds	Probability activity (Pa)	Probability inactivity (Pi)
Hesperidin	0.650	0.006
Diosmin	0.652	0.006
Isorhoirfolin	0.664	0.006
Neodiosmin	0.659	0.006
Poncirin	0.655	0.006
Narirutin	0.662	0.006
Eriocitrin	0.662	0.006
Neohesperidin	0.614	0.008
Tangeretin	0.301	0.060
Nobiletin	0.290	0.064
Sinensetin	0.268	0.073
Heptamethoxyflavone	0.319	0.054
Penicillin (control positive)	0.674	0.005

The results of the interaction analysis between flavonoid compounds in the lime peel (Citrus aurantifolia (Christm.) Swingle) and the protein in MRSA bacteria through STITCH are presented in Figure 1, while the prediction results of penicillin compounds and MRSA proteins are presented in Figure 2.

Fig. 1: Interaction of flavonoids in MeOH:DMSO (1:1, v/v) extract of lime peel with MRSA bacteria

Figure 1 showed the interaction between proteins in MRSA bacteria cells targeted by flavonoids in MeOH: DMSO (1:1, v/v) extract of lime peels. There is an interrelated interaction between SucD and citZ which is an enzyme that helps the synthesis process. SA0703 showed a negative interaction between SucD, SAV1788, and citZ proteins. The computational functional pair prediction showed that flavonoids interact/bind to the AV0703, SAV1788, and SAV2200 proteins.

Fig. 2: Penicillin interactions with MRSA bacteria

Figure 2 showed the interactions between penicillins. There are bpb-related proteins, i.e pbp 3 and pbp 1, which both have primary transpeptidase activity. In the computational prediction of functional paird, it shows that penicillin interact/binds with the pbp, pbp3, pbp1, SAV1112, tst, coa, blah, codY, and met G proteins.

The effect of eight varied concentrations of MeOH: DMSO (1:1, v/v) extract of lime peel on the growth of MRSA bacteria is shown in Figure 3.

(a) (b)

Fig. 3: The inhibitory zone is formed around the well shown by the arrow.

(a) Penicillin (positive control); (b) MeOH: DMSO (1:1, v/v) extract of lime peel of 100 ppm

The results of the observation of antibacterial activity of MeOH: DMSO (1:1, v/v) extract of lime peel with agar well diffusion method after incubation for 24 hours at 37^oC with three repetitions for each concentration variation are presented in Table 3.

Table 3: Mean ± Standard Deviation data of inhibition zone diameter of MeOH: DMSO (1:1, v/v) extract of lime peel against MRSA

Concentrations (ppm)	Mean Diameter of Inhibitory Zones (mm)	Categories of Inhibitory Growth Response
C₀	0 ± 0	None
C₁	17.85 ± 0.07^a	Resistant
C₂	1.79 ± 0.26^e	Less effective
C₃	2.05 ± 0.03^d,e	Less effective
C₄	2.42 ± 0.08^d	Less effective
C₅	5.65 ± 0.52^c	Less effective
C₆	11.61 ± 0.48^b	Weak activity
C₇	12.56 ± 0.37^b	Weak activity
C₈	12.82 ± 0.10^b	Weak activity
C₉	12.96 ± 0.10^b	Weak activity

Notes: Control: C₀ = negative control; C₁ = positive control (Penicillin-G 10 U).

MeOH: DMSO (1:1, v/v) extract of lime peel: C₂= 6.25 ppm; C₃= 12.5 ppm;

C₄= 25 ppm; C₅= 50 ppm; C₆= 100 ppm. Penicillin: resistant= <28 mm;

susceptible= >29 mm.²⁷ Extract from natural ingredients: Strong activity= >20 mm;

Moderate activity: 16-20 mm; Weak activity: 10-15 mm, Less effective= <10 mm.²⁸

The mean value followed by different letters shows a significant difference based on the Duncan test at the 5% level.

Above data were analyzed by one-way ANOVA analysis, provided that the data must be normally distributed and homogeneous in variety. The normality test showed a significance value of 0.866 and the homogeneity of the data showed a significance of 0.092, which means that the data were normally distributed and homogeneous, so that further testing with one-way ANOVA can be carried out.

The results of one-way ANOVA analysis in Table 3 show a significance value of 0.000 which is less than 0.05, indicating a significant difference in the treatment with five variations in the concentration of MeOH : DMSO (1:1) extract of lime peel on MRSA. To determine the concentration of extract which had a significant and insignificant effect on MRSA growth, a further test was carried out with the Duncan test. The Duncan test was used to compare all pairs of treatment averages after the Analysis of variance test was performed.

Table 3 shows the widest diameter of the inhibition zone, which is at the concentration of MeOH: DMSO (1:1, v/v) fraction extract of lime peel of 800ppm with a diameter of the inhibition zone of 12.96mm. The Duncan test showed that the concentration of 200, 400, and 800ppm resulted in not significant difference, however, with concentration of 100ppm there was a significant difference. The concentration of 100, 400, 200, and 100ppm differed significantly from the concentrations of 50, 25, 12.5, and 6.25ppm. Meanwhile, the concentrations of 6.25, 12.5, and 25ppm showed no significant difference.

DISCUSSION:

There is numerous reports on the phytochemical screening antimicrobial activity of different extract in different regions of the world. Such as side effects and resistance of pathogenic microorganisms to antibiotics, in recent time much attention has been compensated to extracts and geologically active compounds isolated from plant species which are used in herbal medicine.³⁰ Knowing the value of bioactive compounds of plants, as well as to get over the multidrug-resistant problem, research institutes and multinational drug companies pay their attention in isolating effective drugs.³¹

The study about presence of phytochemicals in plants considers as active medicinal constituents. Important medicinal phytochemicals such as terpenoids, phenol, flavonoids, carbohydrate, protein, alkaloids, and tannins were present in the studied plants.³² Flavonoids can be classified into flavanones, flavones, and flavonols.^25,33 The composition of flavonoids in lime peel consists of, among others, eriocitrin, narirutin, hesperidin, neohesperidin, neoponcirin, poncirin, isorhoifolin, diosmin, neodiosmin, sinensetin, nobiletin, and tangeretin. The largest component in lime peel is hesperidin.²⁵

Phytochemical screening of medicinal plants is very important in identifying new sources of therapeutically and industrially important compounds. It is imperative to initiate urgent steps for screening of plants for secondary metabolites.³⁴

The interpretation of the PASS SERVER prediction results is a follows: (i) only compounds with a Pa value > Pi have the possibility of being a good compound; (ii) if the Pa value >0.7 then the probability of the compounds activity is experimentally high; (iii) if a value of 0.5< Pa < 0.7, then possibility of the compounds activity being experimentally low and not balanced with known drugs, and (iv) if Pa < 0.5, then the probability of the compounds activity is very low.³⁵

The increasing failure of antibiotic resistance exhibited by pathogenic microbial infectious agents has led to the screening of several medicinal plants for their potential antimicrobial activity.⁹ The antibacterial activity of flavonoids can be carried out in three ways, ie. directly killing bacteria, activating antibiotics synergistically and weakening pathogenic bacteria.³⁶ It is also worth mentioning that flavonoids have exhibited inhibitory activity against the MRSA efflux pump³⁷, and also inhibited peptidoglycan and ribosome synthesis in amoxicillin-resistant Escherichia coli cells.³⁸ They also exhibit inhibitory activity against various types of lactamases produced by bacteria which are key enzymes in deactivating common antibiotics.³⁹

Biologically active compounds present in the plants have always been of great interest. Several previous studies have shown that active compounds in plants can inhibit Staphylococcus aureus bacteria. Plants such as Garcinia mangostana, Zanthoxylum clavaherculis, Cinnamomum zeylanicum, Syzygium aromaticum, Cuminum cyminum.⁴⁰ In another study, Kiruthika et al.⁴¹found inhibitory activity of fresh and dry flower extract of Quisqualis indica against Methicillin-Resistant Staphylococcus aureus. From various studies, it is clear thet the chemical structures of the plants belong to the most commonly encountered classes of higher plant secondary metabolites.¹⁵

Antibacterial activity test of MeOH: DMSO (1:1) fraction of lime peel against Methicillin-Resistant Staphylococcus aureus (MRSA) in vitro used penicillin-G as a positive control. The control was found to produce the inhibition zone diameter of 17.85 mm in resistant category. Penicillin-G can inhibit the growth of Methicillin-Resistant Staphylococcus aureus (MRSA) bacteria even though it is categorized as resistant, because it is possible that in this bacteria there is no gene that encodes resistance against penicillin antibiotic, the blaZ gene.⁴²

Potential antibacterial test of the MeOH:DMSO (1:1, v/v) fraction extract lime peel in vitro in this study was carried out starting from the lowest concentration, 6.25 ppm, to the highest concentration, 100ppm, with the diameter of the inhibition zone none to weak category. This is because the expression of penicillin binding protein (PBP2a) has a low affinity so that it cannot bind to flavonoid compounds in extract. Therefore, peptidoglycan biosynthesis continues.⁴³ Based on the results of this study, it is necessary to carry out an antibacterial combination, namely two or more antibacterial that are used simultaneously and mutually influence the work of each antibacterial. The extracts of several plants that were put together had greater inhibition against bacterial growth than the single plant extracts.^44,45

Utilization of the antibacterial potential of plants is further developed wiil lead to the development of phylomedics against microbes. Antibacterial plant-based materials have enormous therapeutic potential cause they have lower side effects when compared to synthetic antibacterials.⁴⁶

Staphylococcus aureus causes cellulitis, folliculitis, and impetigo. If S. aureus can reach the bloodstream and end up in many different body sites, causes wound infections, abscesses, osteomyelitis, endocarditis, and pneumonia. The correct formulation of antibacterial agents can fight infections caused by S. aureus, thereby saving a person's life.³

The resistance mechanism of Methicillin-Resistant Staphylococcus aureus (MRSA) can occur through the formation of another modified Penicillin Binding protein (PBP), the PBP2a. PBP2a protein expression occurs due to the presence of the genetic element Staphylococcal Cassete Chromosome mec (SCCmec) which carries the mecA gene encoding PBP2a.⁴⁷ The gene encoding the resistance trait of Staphylococcus aureus to methicillin has been identified and characterized, ie. the mecA (gene coding for resistance to methicillin/oxacillin).⁴⁸

CONCLUSION:

The MeOH: DMSO (1:1, v/v) extract of lime peel in concentrations of 100 ppm effectively the inhibited the growth of Methicillin-Resistant Staphylococcus aureus with zone diameter area belonged to weak category.

ACKNOWLEDGEMENT:

The author would like to thank Organic Chemistry Laboratory, FST, Universitas Airlangga and Faculty of Health Science, Universitas Maarif Hasyim Latif.

CONFLICT OF INTEREST:

None to declare.

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Received on 28.03.2021 Modified on 19.09.2021

Research J. Pharm. and Tech. 2022; 15(7):3002-3008.

DOI: 10.52711/0974-360X.2022.00501