Antimicrobial Potential Activity of Extract Selaginella plana (Desv.

Ex Poir.) Hieron against the Growth of Staphylococcus aureus ATCC

25922 and Methicillin-Resistance Staphylococcus aureus (MRSA)

Juen Carla Warella

, Agung Dwi Wahyu Widodo

, Rebekah Juniati Setiabudi

Retno Indrawati Roestamadji

, Maftuchah Rochmanti

and Pudji Lestari

Basic Medical Science Study Program, Faculty of Medicine, Universitas Airlangga, Surabaya, Indonesia

Department of Medical Microbiology, Faculty of Medicine, Universitas Airlangga, Surabaya, Indonesia

Department of Oral Biology, Faculty of Dental Medicine, Universitas Airlangga, Surabaya, Indonesia

Department of Pharmacology, Faculty of Medicine, Universitas Airlangga, Surabaya, Indonesia

Department of Public Health, Faculty of Medicine, Universitas Airlangga, Surabaya, Indonesia.

Keywords: Antimicrobial, Selaginella plana, Staphylococcus aureus, MRSA.

Abstract: For thousands years, medicinal plants have been used as a source of powerful therapeutic agents, and until

now, many medicines are used from natural products derived from plants or their derivatives. Plants that

contain secondary metabolites can be used as antimicrobials, one of them is Selaginella plana. In this study,

there were 8 treatments consisting of 6 treatments of extract concentration, 1 positive control (vancomycin),

and 1 negative control (distilled water) with 3 replications. The antimicrobial test used was the Tube Dilution

method using Mueller Hinton Broth to determine the MIC and Mueller Hinton Agar to determine the MBC.

Selaginella plana extract showed inhibition against Staphylococcus aureus with MIC values of 12.5% and in

MRSA with MIC value of 50%. In MBC test, the killing power of Selaginella plana extract against

Staphylococcus aureus obtained MBC value of 12.5%. Meanwhile, MRSA bacteria showed negative results,

which were indicated by the growth of colonies. Selaginella plana extract (Desv.ex Poir.) Hieron was able to

show antimicrobial activity on Staphylococcus aureus with the MIC value of 12.5%, and the MBC value of

12.5% while in MRSA, Selaginella plana extract (Desv.ex Poir.) Hieron had an MIC value of 50 %, and the

MBC value was negative.

1 INTRODUCTION

For thousands years, plants have been used as

efficacious therapeutic agents, and until recently,

many medicines are used from natural products from

plants and its derivatives (Kinghorn et al., 2011;

Newman and Cragg, 2012). Almost all ancient

findings regarding medicines are sourced from

natural ingredients (Quiason, 2011). WHO report in

2014 recorded that in 129 countries and 80%

population, natural ingredients were used to meet

https://orcid.org/0000-0003-2341-521X

https://orcid.org/0000-0002-8538-719X

https://orcid.org/0000-0003-2171-8743

https://orcid.org/0000-0002-4597-6782

https://orcid.org/0000-0002-9222-9376

https://orcid.org/0000-0003-4725-4676

treatment needs. Similarly, traditional medicines in

China contributed approximately 18% of all

treatments (WHO, 2014).

It was also discovered that more than a third of

medicines (39.1%) authorized by the Food and Drug

Administration (FDA) were sourced from natural

ingredients (Boy et al., 2018). One of the

continuously developed natural molecules is

secondary metabolite substances, in which

approximately 12,000 have been isolated, and the

estimated number is less than 10% (Cowan, 1999).

Warella, J., Wahyu Widodo, A., Setiabudi, R., Roestamadji, R., Rochmanti, M. and Lestari, P.

Antimicrobial Potential Activity of Extract Selaginella plana (Desv. Ex Poir.) Hieron against the Growth of Staphylococcus aureus ATCC 25922 and Methicillin-Resistance Staphylococcus

aureus (MRSA).

DOI: 10.5220/0010490802450253

In Proceedings of the 1st Jenderal Soedirman International Medical Conference in conjunction with the 5th Annual Scientiﬁc Meeting (Temilnas) Consortium of Biomedical Science Indonesia

(JIMC 2020), pages 245-253

ISBN: 978-989-758-499-2

245

In Indonesia, the utilization of plant-based

medicines is a part of national cultivation and has

been existing for centuries. However, its

effectiveness and safety have not been supported by a

comprehensive study (WHO, 2010). One of the

biological resources in Indonesia is Selaginella Pal.

Beauv (Selaginellaceae Reichb). Selaginella has been

used as an alternative medicine in several traditional

treatments, such as to cure injuries, skin diseases,

cancers (Chen et al., 2005), anti-inflammation (Raj et

al., 2006; Won et al., 2006), rheumatic, and as anti-

microbes.

Plants with antimicrobial potential commonly

have secondary metabolites. Selaginella has species-

dependent molecular bioactivities, such as phenolic

(flavonoid), alkaloid, and terpenoid contents.

However, bioflavonoids (a dimeric form of

flavonoids) are the key bioactive substances of

Selaginella, consisting of 13 substances, particularly

amentoflavone and ginkgetin (Setyawan, 2011).

Antimicrobial substances can be used as a strategy to

tackle health problems related to bacteria, fungi, and

parasites.

According to (WHO, 2015), one of the current

global problems is antimicrobial resistance

threatening public health. Hence, the search for

effective antimicrobial agents can help prevent and

heal the patients. Antimicrobial agent from natural

substances is one of the alternative treatments that is

continuously developed. The antimicrobial agent is

classified into six categories, namely biosynthesis,

biological source, biological function, molecular

properties, structure, composition, and molecular

purpose (Castro-rosas et al., 2017)

From previous studies, it is discovered that only

several species had been observed in detail, such as

Selaginella uncinata (Zou et al., 2013b; Zou et al.,

2014; Taylor et al., 2013; Zou et al., 2016b),

Selaginella doederleinii (Li et al., 2016), Selaginella

involvens (Long et al., 2015), Selaginella tamariscina

(Xu, et a.l, 2011a; Xu et al., 2011b; Xu et al., 2015ab),

Selaginella moellendorffii (Zou et al., 2016a; Zeng et

al., 2017; Zou et al., 2013a), and Selaginella

willdenowii (Chai and Wong, 2012). Meanwhile, the

most distributed Selaginella in Indonesia, i.e.,

Selaginella plana is yet to be observed further.

Based on the background, the authors were

interested in conducting a study regarding the

“Antimicrobial Potential Activity of Extract

Selaginella plana (Desv. Ex Poir.) Hieron against the

Growth of Staphylococcus aureus ATCC 25922 and

Methicillin-Resistance Staphylococcus aureus

(MRSA)”.

2 MATERIAL AND METHODS

In this section, the authors explain the steps in testing

the potential antimicrobial activity of Selaginella

plana (Desv. ex poir.) Hieron extract against the

growth of Staphylococcus aureus ATCC 25922 and

methicillin-resistance Staphylococcus aureus

(MRSA). This study was conducted in the

Pharmacology Laboratory, Faculty of Medicine,

Universitas Airlangga and Medical Microbiology

Laboratory, Faculty of Medicine, Universitas

Airlangga since 15 February 2020 to 14 March 2020.

2.1 Materials

The primary material used was Selaginella plana.

The solvent used in the extraction process was

ethanol 96%. Antimicrobial materials consisted of

Mueller Hinton Agar, Mueller Hinton broth,

vancomycin, test bacteria Staphylococcus aureus

ATCC 25922, methicillin-resistance Staphylococcus

aureus (MRSA), suspension of 0.5 McFarland, and

distilled water.

Equipment used were autoclave GEA FSF-24LDJ

(Hahei, China), oven, refrigerator LG GN-

B215SQMT (Taizhou, China), vortex GEMMY VM-

300 (Taiwan), incubator Memmert UN 33 53L

(Germany), vacuum rotary evaporator Heidolph VV

2000 (Nuremberg, Germany), digital scale FX-300i

(Max 320 g), micropipettes, Bunsen, smear loops, and

glass equipment such as test tubes, petri dish,

Erlenmeyer flask, beaker glass, and volumetric

pipettes.

2.2 Plant Extraction Preparation

In this study, Selaginella plana obtained from Kairatu

Village, West Seram, Maluku Province, on 10

February 2020.

As many as 1 kg of Selaginella plana leaves was

washed using running water, dried under shades,

chopped to pieces, and dried in the oven. Dried leaves

were blended into powders of 600 g. The extraction

process used a maceration method with ethanol 96%

as a solvent. The 600 g powder was soaked in ethanol

of 5400 ml while stirred for 24 hours. The top layer

was taken using Whatman paper no. 41, and the

soaking process was repeated for three times. The

filtrate was dried in the rotary evaporator of 60°C

until the ethanol solution was separated from the

active substance.

The extract was weighed and calculated using the

following formula: Extract % = dried mass / extract

JIMC 2020 - 1’s t Jenderal Soedirman International Medical Conference (JIMC) in conjunction with the Annual Scientiﬁc Meeting

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volume x 1000 ml. The maceration process resulted

in Selaginella plana extracts of 70 g.

2.3 Antimicrobial Activity of the Plant

Extracts

This study used Tube Dilution Test method. This

method was utilized to determine the MIC (Minimum

Inhibitory Concentration) and MBC (Minimum

Bactericidal Concentration). The dilution method

testing was carried out according to the

recommendation of the Clinical and Laboratory

Standards Institute for the determination of MIC and

MBC.

2.3.1 Bacterial Strain

The antimicrobial activity testing of Selaginella

plana extracts used two bacteria strains, i.e.,

Staphylococcus aureus ATCC 25922 and methicillin-

resistance Staphylococcus aureus (MRSA). The

Staphylococcus aureus ATCC 25922 bacteria strain

was obtained from the Health Laboratory Center

Surabaya, and the methicillin-resistance

Staphylococcus aureus (MRSA) bacteria strain was

obtained from the Microbiology Laboratory, Faculty

of Medicine, Universitas Airlangga, Surabaya.

2.3.2 Preparation of Bacterial Suspension

The bacteria rejuvenation process used Mueller

Hinton Agar. The incubation was carried out for 24

hours at the optimum temperature of 37°C. The

bacteria suspension production used Mueller Hinton

broth. One smear of microbes was put into 5 ml media

in the test tube, vortexed, and adjusted to the standard

of 0.5 McFarland (1.5 x 10

CFU/ml).

2.3.3 Antimicrobial Activity Assay

There were 6 Selaginella plana (Desv.ex Poir.)

Hieron extracts’ concentrations including 100%,

50%, 25%, 12.5%, 6.25%, and 3.125%. One positive

control used Vancomycin 30 mg, and 1 negative

control used distilled water. The dilution process was

conducted in stages, initiated by the treatment 1 (P1)

group by putting 1 ml of 100% Selaginella plana

extract into 1 ml of Mueller Hinton broth and

vortexed them to be mixed. The treatment 2 (P2)

group was made by putting 1 ml of 50% P1 solution

into 1 ml of Mueller Hinton broth and vortexed them

to be mixed. The same step was applied to P3 group

of 25% sample concentration, the P4 group of 1.25%,

the P5 group of 6.25%, and the P6 group of 3.125%.

Each group was added with 1 ml of bacteria

suspension (1.5 x 10

CFU/ml) and repeated three

times. Incubations were carried out for 24 hours and

72 hours with a temperature of 37°C in incubators,

which were then observed and compared with the

positive and negative controls.

2.3.4 Determination of Minimum Inhibitory

Concentration (MIC)

Minimum Inhibitory Concentration (MIC) is the

minimum extract concentration to inhibit microbial

growth after being incubated for 24 hours. The

determination of Minimum Inhibitory Concentration

(MIC) was conducted by taking all incubated

treatment groups, vortexing each tube of different

concentrations, and observing the smallest

concentration to inhibit bacterial growth (visually

marked by three observers) and determined as the

MIC (Brantner and Grein, 1994; Chérigo et al., 2009).

2.3.5 Determination of Minimum

Bactericidal Concentration (MBC)

Minimum Bactericidal Concentration (MBC) is the

minimum concentration of test materials to kill

bacteria, measured using the colony counter. The

Minimum Bactericidal Concentration (MBC) was

conducted by taking samples and smearing them to

the Mueller Hinton agar and incubated at 37°C for 24

hours. It was then determined for the smallest

concentration where microbial colonies stopped

growing on the media.

The colony growth on the Mueller Hinton agar

was declared with: (-) if more than 10 colonies were

obtained on the Petri dish, (+) if less than 10 colonies

were obtained on the Petri dish, and if colonies were

grouping, it was counted as one colony.

3 RESULTS

3.1 Extraction

Selaginella plana with a wet weight of 1 kg was dried

to obtain a dry weight of 600 grams. Selaginella

plana was then extracted with a maceration method

using ethanol 96% solvent. Extracts from the

maceration process were 70 grams.

3.2 Minimum Inhibitory Concentration

(MIC) of the Plant Extract

The microbial activity test was conducted using broth

dilution method. Concentrations used were 100%,

Antimicrobial Potential Activity of Extract Selaginella plana (Desv. Ex Poir.) Hieron against the Growth of Staphylococcus aureus ATCC

25922 and Methicillin-Resistance Staphylococcus aureus (MRSA)

247

50%, 25%, 12.5%, 6.25%, and 3.125%. The negative

control was distilled water, while the positive control

was vancomycin.

The test results of Minimum Inhibitory

Concentration (MIC) of Selaginella plana (desv.ex

poir.)

Hieron extracts are presented in Table 1. The

Table 1: Minimum Inhibitory Concentration of Selaginella plana.

Bacterial Strain Replication

Test Concentration

100% 50% 25% 12.5% 6.25% 3.125%

Staphylococcus aureus

ATCC 25922

1 + + + + + -

2 + + + + - -

3 + + + + - -

methicillin-resistant

Staphylococcus aureus (MRSA)

1 + + + - - -

2 + + - - - -

3 + + - - - -

results of antimicrobial activity testing showed

turbidity differences on different concentration

levels. Therefore, the Minimum Inhibitory

Concentration (MIC) on a particular concentration

was determined.

Figure 1: The Minimum Inhibitory Concentration (MIC) of

Staphylococcus aureus ATCC 25922.

Figure 2: The Minimum Inhibitory Concentration (MIC) of

methicillin-resistance Staphylococcus aureus (MRSA).

The testing of Selaginella plana extract on

Staphylococcus aureus ATCC 25922 bacteria was

conducted on different concentrations, i.e., 100%,

50%, 25%, 12.5%, 6.25%, and 3.125%. The results in

Table 1 present that the first (100%), second (50%),

third (25%), and fourth (12.5%) tubes showed no

turbidity. Therefore, the fourth (12.5%) tube was

determined as the Minimum Inhibitory

Concentration. On the positive control tube with

vancomycin, no turbidity presented. Meanwhile, the

negative control tube with distilled water showed

turbidity (Figure 1).

The testing for MIC was also applied to the

methicillin-resistance Staphylococcus aureus

(MRSA) bacteria with the same concentration of

100%, 50%, 25%, 12.5%, 6.25%, and 3.125%. The

results in Table 1 show that the 100% and 50%

concentrations had abilities to inhibit MRSA’s

growth, marked by no turbidity in tubes. Therefore,

the 50% concentration was considered as the MIC.

However, the inhibitory potential of Selaginella

plana extract was considered weak because the lower

concentrations of 25%, 12.5%, 6.25%, and 3.125%

showed turbidity and thick lumps. The positive

control tube with vancomycin showed no turbidity,

and the negative control tube with distilled water

showed turbidity (Figure 2).

However, due to the incomplete screening of

Selaginella plana extraction results, it may leave

dregs that pose bias in determining the MIC.

Therefore, the microbes’ growth inhibition was also

tested using selective growth media for each microbe.

It aimed to confirm the presence or absence of

microbes’ growth in a particular concentration

showing the Minimum Inhibitory Concentration

(MIC). The result obtained was determined as the

Minimum Bactericidal Concentration (MBC).

JIMC 2020 - 1’s t Jenderal Soedirman International Medical Conference (JIMC) in conjunction with the Annual Scientiﬁc Meeting

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3.3 The Minimum Bactericidal

Concentration (MBC) of the Plant

Extract

The test results of Minimum Bactericidal

Concentration (MBC) of Selaginella plana (desv.ex

poir.) Hieron extracts are presented in Table 2.

The Minimum Bactericidal Concentration (MBC)

test to Staphylococcus aureus ATCC 25922 bacteria

on the concentrations of 100%, 50%, 25%, and 12.5%

showed positive results, marked with zero growth of

Staphylococcus aureus ATCC 25922 colony on all

test concentrations (Figure 3).

The Minimum Bactericidal Concentration (MBC)

test to MRSA bacteria on the concentrations of 100%

and 50% showed negative results, marked with

MRSA colony growth on all concentrations. It shows

that Selaginella plana extracts are incapable to kill

MRSA (Figure 4).

Table 2: Minimum Bactericidal Concentration of Selaginella plana.

Bacterial Strain Replication

Test Concentration

100% 50% 25% 12.5% 6.25% 3.125%

Staphylococcus aureus

ATCC 25922

1 + + + + + -

2 + + + + - -

3 + + + + - -

methicillin-resistant

Staphylococcus aureus

(MRSA)

1 +

- - - - -

2 - - - - - -

3 - - - - - -

Figure 3: Minimum Bactericidal Concentration (MBC) of

Staphylococcus aureus ATCC 25922.

Figure 4: Minimum Bactericidal Concentration (MBC) of

and methicillin-Resistance Staphylococcus aureus (MRSA)

4 DISCUSSION

4.1 Minimum Inhibitory Concentration

(MIC) of Selaginella plana

Staphylococcus aureus and methicillin-resistance

Staphylococcus aureus (MRSA) bacteria are

pathogenic microorganisms commonly infecting

humans, and many researchers suggested that these

microbes are resistant to medicines (Kumar, 2016;

Passàli et al., 2007; Ksiezopolska, 2018; Onanuga,

2011). It was discovered that secondary metabolite

substances in plants play a vital role as antimicrobial,

especially phenolic (Gechev et al., 2014).

On the MRSA bacteria, the MIC test showed

positive results, marked by the absence of turbidity in

a high concentration. The previous study discovered

that a flavonoid substance of 7-O-Butyl Naringenin

had activity against MRSA strains on a lower MIC

than natural flavonoids (Lee et al., 2013a). Glabrol

elements in flavonoids disturb membrane potentials

and permeability, hence, potential to be used as an

anti-microbe against MRSA (Wu et al., 2019).

The study by (Cao et al., 2010b) on active

substances in Selaginella pulvinata have good and

significant inhibitory activity against Staphylococcus

aureus with a MIC value of 9.6 μg/ml. According to

(Zou et al., 2016a), flavonoid compounds can inhibit

S.aureus growth with a MIC value of 12.5 μg/ml.

Flavonoids ability as anti-microbes depends on

the aromatic ring structure (Xie et al., 2014).

Antimicrobial Potential Activity of Extract Selaginella plana (Desv. Ex Poir.) Hieron against the Growth of Staphylococcus aureus ATCC

25922 and Methicillin-Resistance Staphylococcus aureus (MRSA)

249

Flavonoid activities disturb membrane integrity due

to an interaction with phospholipids that change the

membrane protein’s structure and function, adhere to

the membrane’s hydrophobic and hydrophilic sides,

and cause dysfunction of plasm membrane’s works

(Górniak et al., 2019). It also causes cell agglutination

(Babii et al., 2016), energy metabolism disruption,

nucleic acid synthesis, coenzyme metabolism, and

cell leaking (Cushnie and Lamb, 2011).

Bacteria used in this study were gram-positive

bacteria, i.e., Staphylococcus aureus and methicillin-

resistance Staphylococcus aureus (MRSA), which are

also influenced by flavonoids. The reason is because

positive gram bacteria cell’s walls contain a high

amount of peptidoglycans. On the outer cell part,

phosphate groups contain ≥ 80% negative charges

(Cha et al., 2006). It causes interaction between

negative and positive charges on the carbon atom of

1.3-dithiolium flavonoid ring (Bahrin et al., 2014). As

a result, an intracellular leak. Another study also

found that a particular dosage of saponins was

effective in damaging Staphylococcus aureus cell

walls (Khan et al., 2018). However, terpenoids do not

pose activities on Staphylococcus aureus, while on

methicillin-resistance Staphylococcus aureus

(MRSA), terpenoids posed activities as anti-MRSA,

although not effective as standard medicines

(Nzogong et al., 2018)

4.2 Minimum Bactericidal

Concentration (MBC) of

Selaginella plana

The MBC value test in Table 2 shows that Selaginella

plana extracts on the concentrations of 100%, 50%,

25%, 12.5%, and 6.25% had positive results against

Staphylococcus aureus.

Different results were presented by methicillin-

resistance Staphylococcus aureus (MRSA) bacteria.

Selaginella plana extracts had no ability as

bactericidal, marked by bacteria colony growth on

Petri dishes. Phytochemical substances such as tannin

and polyphenol are major contributors to inhibit

methicillin-resistance Staphylococcus aureus

(MRSA) bacteria. Therefore, these substances'

absence affects the non-synergized multi-target

effects against methicillin-resistance Staphylococcus

aureus (MRSA) bacteria (Chew et al., 2018). The

mixture of constituents may act on several

antibacterial targets concurrently, i.e. depolarizing

the cell membrane, inhibiting the efflux pump,

disintegrating the genetic materials (Coutinho et al.,

2009; Efferth and Koch, 2010).

A study conducted by (Chew et al., 2018) found

that tannins in plants could contribute to MRSA

inhibitory activity. The potency of the phytochemical

compound can be increased if it is combined with

other medicines since it has different targets in

MRSA. Phytochemical compounds can change the

permeability of the outer cell membrane, inhibit the

efflux pump, change the active site, and β-lactamase

inhibitors (Kubo et al., 2003).

Multi-target effects of phytochemical substances

are known to act as anti-MRSA by depolarizing cell

membranes, inhibiting efflux pumps, and damaging

genetic materials (Coutinho et al., 2009; Efferth and

Koch, 2010). Methicillin-resistant Staphylococcus

aureus (MRSA) resistance towards extracts is caused

by mucosa layer thickness surrounding cell walls.

The cell wall layer produced by resistant isolates is

thicker than the sensitive walls of strains (Amira,

2016). It was caused by the decreased penicillin-

binding proteins (PBP) activity affecting the cross-

link in peptidoglycan and an increase in gene

expression related to cell wall synthesis caused an

increase in the production of teichoic acid in the cell

wall (García, et al., 2017).

A quantitative study against the inhibitory of

phytochemical compounds needs to be conducted in

determining Minimum Inhibitory Concentration

(MIC) and Minimum Bactericidal Concentration

(MBC) by observing the Optical Density (OD) value

in each tested concentration and further investigation

is conducted against the specific phytochemical

compound in preventing, inhibiting, and degrading

the biofilm growth in each microbe.

5 CONCLUSION

Based on the study results, conclusions can be drawn

as follow:

1. Selaginella plana (Desv.ex Poir.) Hieron extracts

have the potential as anti-microbes on the

Minimum Inhibitory Concentration (MIC) test

with the concentrations of 100%, 50%, 25%, and

12.5% could inhibit Staphylococcus aureus’

growth, and the concentrations of 100% and 50%

can inhibit MRSA’s growth.

2. Selaginella plana (Desv.ex Poir.) Hieron extracts

have the potential as bactericidal on the Minimum

Bactericidal Concentration (MBC) test with the

concentrations of 100%, 50%, 25%, and 12.5%

can kill Staphylococcus aureus’ growth.

However, the results are negative against MRSA

with colony growth on the concentrations of

100% and 50%.

JIMC 2020 - 1’s t Jenderal Soedirman International Medical Conference (JIMC) in conjunction with the Annual Scientiﬁc Meeting

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250

REFERENCES

Amira, H. A. A. A. (2016). Effect of plants extracts on the

growth of Candida albicans and Staphylococcus aureus.

African Journal of Pharmacy and Pharmacology,

10(16), 337–345.

https://doi.org/10.5897/ajpp2016.4522

Babii, C., Bahrin, L. G., Neagu, A. N., Gostin, I., Mihasan,

M., Birsa, L. M., & Stefan, M. (2016). Antibacterial

activity and proposed action mechanism of a new class

of synthetic tricyclic flavonoids. Journal of Applied

Microbiology, 120(3), 630–637.

https://doi.org/10.1111/jam.13048

Bahrin, L. G., Apostu, M. O., Birsa, L. M., & Stefan, M.

(2014). The antibacterial properties of sulfur containing

flavonoids. Bioorganic and Medicinal Chemistry

Letters, 24(10), 2315–2318.

https://doi.org/10.1016/j.bmcl.2014.03.071

Balouiri, M., Sadiki, M., & Ibnsouda, S. K. (2016).

Methods for in vitro evaluating antimicrobial activity :

A review. Journal of Pharmaceutical Analysis, 6(2),

71–79. https://doi.org/10.1016/j.jpha.2015.11.005

Boy, H. I. A., Rutilla, A. J. H., Santos, K. A., Ty, A. M. T.,

Yu, A. I., Mahboob, T., … Nissapatorn, V. (2018).

Recommended Medicinal Plants as Source of Natural

Products: A Review. Digital Chinese Medicine, 1(2),

131–142. https://doi.org/10.1016/s2589-

3777(19)30018-7

Cao, Y., Chen, J., Tan, N., Oberer, L., Wagner, T., Wu, Y.,

… Wang, Q. (2010b). Antimicrobial selaginellin

derivatives from Selaginella pulvinata. Bioorganic &

Medicinal Chemistry Letters, 20(8), 2456–2460.

https://doi.org/10.1016/j.bmcl.2010.03.016

Castro-rosas, J., Ferreira-grosso, C. R., Gómez-aldapa, C.

A., Rangel-vargas, E., Rodríguez-marín, M. L.,

Guzmán-ortiz, F. A., & Falfan-cortes, R. N. (2017).

Recent advances in microencapsulation of natural

sources of antimicrobial compounds used in food - A

review. Food Research International, 102(September),

575–587.

https://doi.org/10.1016/j.foodres.2017.09.054

Cha, T. W., Guo, A., & Zhu, X. Y. (2006). Formation of

supported phospholipid bilayers on molecular surfaces:

Role of surface charge density and electrostatic

interaction. Biophysical Journal, 90(4), 1270–1274.

https://doi.org/10.1529/biophysj.105.061432

Chai, T., & Wong, F. (2012). Antioxidant properties of

aqueous extracts of Selaginella willdenowii. Journal of

Medicinal Plants Research, 6(7), 1289–1296.

https://doi.org/10.5897/JMPR11.1376

Chen, J., Duh, C., & Chen, J. (2005). New Cytotoxic

Biflavonoids from Selaginella delicatula. Planta

Medica, 71(7), 659–665. https://doi.org/10.1055/s-

2005-871273

Chew, Y. L., Mahadi, A. M., Wong, K. M., & Goh, J. K.

(2018). Anti-methicillin-resistance Staphylococcus

aureus (MRSA) compounds from Bauhinia kockiana

Korth. And their mechanism of antibacterial activity.

BMC Complementary and Alternative Medicine, 18(1),

1–9. https://doi.org/10.1186/s12906-018-2137-5

Coutinho, H. D. M., Costa, J. G. M., Lima, E. O., Falcão-

Silva, V. S., & Siqueira, J. P. (2009). Herbal therapy

associated with antibiotic therapy: Potentiation of the

antibiotic activity against methicillin - Resistant

Staphylococcus aureus by Turnera ulmifolia L. BMC

Complementary and Alternative Medicine, 9, 1–4.

https://doi.org/10.1186/1472-6882-9-13

Cowan, M. M. (1999). Plant Products as Antimicrobial

Agents. Clinical Microbiology Reviews, 12(4), 564–

582. https://doi.org/10.1128/CMR.12.4.564

Cushnie, T. P. T., & Lamb, A. J. (2011). Recent advances

in understanding the antibacterial properties of

flavonoids. International Journal of Antimicrobial

Agents, 38(2), 99–107.

https://doi.org/10.1016/j.ijantimicag.2011.02.014

Efferth, T., & Koch, E. (2010). Complex Interactions

between Phytochemicals. The Multi-Target

Therapeutic Concept of Phytotherapy. Current Drug

Targets, 12(1), 122–132.

https://doi.org/10.2174/138945011793591626

García, A. B., Viñuela-Prieto, J. M., López-González, L., &

Candel, F. J. (2017). Correlation between resistance

mechanisms in Staphylococcus aureus and cell wall and

septum thickening. Infection and Drug Resistance, 10,

353–356. https://doi.org/10.2147/IDR.S146748

Gechev, T. S., Hille, J., Woerdenbag, H. J., Benina, M.,

Mehterov, N., Toneva, V., Mueller-roeber, B. (2014).

Natural products from resurrection plants : Potential for

medical applications. Biotechnology Advances, 32(6),

1091–1101.

https://doi.org/10.1016/j.biotechadv.2014.03.005

Górniak, I., Bartoszewski, R., & Króliczewski, J. (2019).

Comprehensive review of antimicrobial activities of

plant flavonoids. Phytochemistry Reviews, 18(1), 241–

272. https://doi.org/10.1007/s11101-018-9591-z

Khan, M. I., Ahhmed, A., Shin, J. H., Baek, J. S., Kim, M.

Y., & Kim, J. D. (2018). Green Tea Seed Isolated

Saponins Exerts Antibacterial Effects against Various

Strains of Gram Positive and Gram Negative Bacteria,

a Comprehensive Study In Vitro and In Vivo. Evidence-

Based Complementary and Alternative Medicine, 2018,

12. https://doi.org/10.1155/2018/3486106

Kinghorn, A. D., Pan, L., Fletcher, J. N., & Chai, H. (2011).

The relevance of higher plants in lead compound

discovery programs. Journal of Natural Products,

74(6), 1539–1555. https://doi.org/10.1021/np200391c

Ksiezopolska, E., & Gabaldón, T. (2018). Evolutionary

emergence of drug resistance in candida opportunistic

pathogens. Genes, 9(9).

https://doi.org/10.3390/genes9090461

Kubo, I., Fujita, K. I., & Nihei, K. I. (2003). Molecular

design of multifunctional antibacterial agents against

methicillin resistant Staphylococcus aureus (MRSA).

Bioorganic and Medicinal Chemistry, 11(19), 4255–

4262. https://doi.org/10.1016/S0968-0896(03)00433-4

Kumar, M. (2016). Multidrug-resistant Staphylococcus

aureus, India, 2013–2015. Emerging Infectious

Diseases, 22(9), 1666–1667.

https://doi.org/10.3201/eid2209.160044

Antimicrobial Potential Activity of Extract Selaginella plana (Desv. Ex Poir.) Hieron against the Growth of Staphylococcus aureus ATCC

25922 and Methicillin-Resistance Staphylococcus aureus (MRSA)

251

Lee, K. A., Moon, S. H., Lee, J. Y., Kim, K. T., Park, Y. S.,

& Paik, H. D. (2013a). Antibacterial activity of a novel

flavonoid, 7-O-butyl naringenin, against methicillin-

resistant Staphylococcus aureus (MRSA). Food

Science and Biotechnology, 22(6), 1725–1728.

https://doi.org/10.1007/s10068-013-0272-9

Li, J., Yu, X., Cao, D., Li, D., Zeng, W., Zhang, G., & Tan,

G. (2016). NU SC. Fitoterapia.

https://doi.org/10.1016/j.fitote.2016.11.014

Long, H., Zou, H., Li, F., Li, J., Luo, P., Zou, Z., & Hu, C.

(2015). Fitoterapia Involven fl avones A – F , six new

fl avonoids with 3 ′ -aryl substituent from Selaginella

involven. Fitoterapia, 105, 254–259.

https://doi.org/10.1016/j.fitote.2015.07.013

Newman, D. J., & Cragg, G. M. (2012). Natural products as

sources of new drugs over the 30 years from 1981 to

2010. Journal of Natural Products, 75(3), 311–335.

https://doi.org/10.1021/np200906s

Nzogong, R. T., Ndjateu, F. S. T., Ekom, S. E., Fosso, J. A.

M., Awouafack, M. D., Tene, M., … Tamokou, J. de D.

(2018). Antimicrobial and antioxidant activities of

triterpenoid and phenolic derivatives from two

Cameroonian Melastomataceae plants: Dissotis

senegambiensis and Amphiblemma monticola. BMC

Complementary and Alternative Medicine, 18(1), 1–11.

https://doi.org/10.1186/s12906-018-2229-2

Onanuga, A., & Temedie, T. C. (2011). Multidrug-resistant

intestinal Staphylococcus aureus among self-medicated

healthy adults in Amassoma, South-South, Nigeria.

Journal of Health, Population and Nutrition, 29(5),

446–453. https://doi.org/10.3329/jhpn.v29i5.8898

Passàli, D., Lauriello, M., Passàli, G. C., Passàli, F. M., &

Bellussi, L. (2007). Group A streptococcus and its

antibiotic resistance. Acta Otorhinolaryngologica

Italica : Organo Ufficiale Della Società Italiana Di

Otorinolaringologia e Chirurgia Cervico-Facciale,

27(1), 27–32. https://doi.org/10.1155/2019/5739247

Quiason, S. (2011). Concise History of Drug Discovery

Drug Discoveries and Invention. A Global

Perspective. 3-23, 154-225.

Raj, Y., Won, J., Young, J., Woo, H., Gwang, H., Woo, E.,

& Wook, K. (2006). Potent inhibition of the inductions

of inducible nitric oxide synthase and cyclooxygenase-

2 by taiwania X avone. 15, 217–225.

https://doi.org/10.1016/j.niox.2006.01.001

Setyawan, A. D., & Darusman, L. K. (2008). REVIEW :

Senyawa Biflavonoid pada Selaginella Pal. Beauv . dan

Pemanfaatannya Review : Biflavonoid compounds of

Selaginella Pal . Beauv. and its benefit. Biodiversitas,

9, 64–81. https://doi.org/10.13057/biodiv/d090115

Taylor, P., Zou, H., Xu, K., Li, F., Zou, Z., Long, H.,Tan,

G. (2013). Journal of Asian Natural Products

Uncinataflavone , a new flavonoid with a methyl

benzoate substituent from Selaginella uncinata.

(March), 37–41.

https://doi.org/10.1080/10286020.2013.771345

Won, J., Raj, Y., Kim, M., Woo, E., Kyoon, H., & Wook,

K. (2006). Inhibition of inducible nitric oxide synthase

by sumaflavone isolated from Selaginella tamariscina.

105, 107–113.

https://doi.org/10.1016/j.jep.2005.10.001

World Health Organization (WHO). (2010). Traditional

Medicine in Republic of Indonesian Traditional

Medicine. 23–36. Retrieved from

http://www.searo.who.int/entity/medicines/topics/tradi

tional_medicines_in_republic_of_indonesia.pdf

World Health Organization (WHO). (2014). WHO

Traditional Medicine Strategy Plan 2014-2020. WHO

Traditional Medicine Strategy, (March 2014), 120–

125. Retrieved from

https://www.who.int/medicines/publications/traditiona

l/trm_strategy14_23/en/

World Health Organization (WHO). (2015). Global Action

Plan on Antimicrobial Resistance. Microbe Magazine,

10(9), 354–355.

https://doi.org/10.1128/microbe.10.354.1

Wu, S., Yang, Z., Liu, F., Peng, W., Qu, S., & Li, Q. (2019).

Antibacterial Effect and Mode of Action of Flavonoids

From Licorice Against Methicillin-Resistant

Staphylococcus aureus. Frontiers in Microbiology,

10(November), 1–14.

https://doi.org/10.3389/fmicb.2019.02489

Xie, Y., Yang, W., Tang, F., Chen, X., & Ren, L. (2014).

Antibacterial Activities of Flavonoids: Structure-

Activity Relationship and Mechanism. Current

Medicinal Chemistry, 22(1), 132–149.

https://doi.org/10.2174/0929867321666140916113443

Xu, K., Zou, H., Tan, Q., Li, F., Liu, J., & Xiang, H.

(2011a). Selaginellins I and J , two new alkynyl phenols

, from Selaginella tamariscina ( Beauv .) Spring. 13(2),

93–96. https://doi.org/10.1080/10286020.2010.536535

Xu, K., Zou, H., Li, F., & Xiang, H. (2011b). Journal of

Asian Natural Products Two new selaginellin

derivatives from Selaginella tamariscina ( Beauv .)

Spring. (December 2014), 37–41.

https://doi.org/10.1080/10286020.2011.558840

Xu, K., Li, J., Zhu, G., He, X., & Li, F. (2015a). Journal of

Asian Natural Products New Selaginellin derivatives

from Selaginella tamariscina. (April), 37–41.

https://doi.org/10.1080/10286020.2015.1016001

Xu, K., Li, J., Zhu, G., He, X., & Li, F. (2015b). New

Selaginellin derivatives from Selaginella tamariscina.

Journal of Asian Natural Products Research, (April),

37–41.

https://doi.org/10.1080/10286020.2015.1016001

Zeng, W., Yao, C., Xu, P., Zhang, G., Liu, Z., Xu, K., …

Tan, G. (2017). A new neolignan from Selaginella

moellendorffii Hieron. Natural Product Research,

6419(March),0.

https://doi.org/10.1080/14786419.2017.1297935

Zou, Z., Xu, K., Li, F., Zou, H., Liu, M., Zhang, Q., … Tan,

G. (2013a). Original article A new pyrrole alkaloid

from Selaginella moellendorfii Hieron. Chinese

Chemical Letters, 24(2), 114–116.

https://doi.org/10.1016/j.cclet.2013.01.028

Zou, H., Xu, K., Zou, Z., Long, H., Li, F., Li, J., … Tan, G.

(2013b). Journal of Asian Natural Products A new

flavonoid with 6-phenyl substituent from Selaginella

JIMC 2020 - 1’s t Jenderal Soedirman International Medical Conference (JIMC) in conjunction with the Annual Scientiﬁc Meeting

(Temilnas) Consortium of Biomedical Science Indonesia (KIBI )

252

uncinata. (October), 37–41.

https://doi.org/10.1080/10286020.2012.745515

Zou, H., Xu, K., Li, F., Zou, Z., Liu, R., Liu, R., … Tan, G.

(2014). Fitoterapia Uncifl avones A – F , six novel fl

avonoids from Selaginella uncinata ( Desv .) Spring.

Fitoterapia, 99, 328–333.

https://doi.org/10.1016/j.fitote.2014.10.012

Zou, Z., Xu, P., Wu, C., Zhu, W., Zhu, G., He, X., … Tan,

G. (2016a). Fitoterapia., Carboxymethyl fl avonoids

and a chromone with antimicrobial activity from

Selaginella moellendorffii Hieron. 111, 124–129.

https://doi.org/10.1016/j.fitote.2016.04.022

Zou, H., Xu, P., iu, R., Zou, Z., Li, J., Zhong, A., & Hu, J.

(2016b). Selayclicbiflavone A , an unusual macrocyclic

biflavone from Selaginella uncinata (Desv .) Spring.

Tetrahedron Letters, 37, 37–39.

https://doi.org/10.1016/j.tetlet.2016.01.038

Antimicrobial Potential Activity of Extract Selaginella plana (Desv. Ex Poir.) Hieron against the Growth of Staphylococcus aureus ATCC

25922 and Methicillin-Resistance Staphylococcus aureus (MRSA)

253