Antibacterial Activity of Methanol Extracts and Compounds of Wualae

(Etlingera elatior) Fruits from Southeast Sulawesi-Indonesia

I. Sahidin,

Syefira Salsabila

, W. Wahyuni

, M. Hajrul Malaka

, Imran

and Marianti A. Manggau

Faculty of Pharmacy, Universitas Halu Oleo Kendari 93232 Southeast Sulawesi, INDONESIA

Department of Chemistry, Faculty of Mathematics and Natural Sciences Universitas Halu Oleo Kendari 93232 Southeast

Sulawesi, INDONESIA

Pharmacology, Faculty of Pharmacy, Universitas Hasanuddin, Makassar 90245, South Sulawesi, INDONESIA

Keywords: Etlingera elatior, fruits, vanilic acid, p-hydroxybenzoic acid and antibacteria

Abstract: Wualae (Tolakinese) or Etlingera elatior grows bulky in Southeast Sulawesi. The fruit of this plant is

widely used as cooking spices and traditional medicine. Scientific studies of the fruit and its properties

against certain diseases are still very limited. The aim of the article shares the chemical content of E.

elatior fruits and its activity against various selected pathogenic bacteria. Isolation was performed by

chromatographic methods, including Thin Layer Chromatography (TLC), Vacuum Liquid

Chromatography (VLC), and Radial Chromatography (RC). Structure of the isolated compounds was

elucidated by using spectroscopic techniques, i.e. IR and NMR-1D spectroscopy (

H and

C-NMR) and

comparing with similar data from the literature. The activity of the methanol extracts and the isolated

compounds were evaluated against bacteria using the diffusion agar method. The tested bacteria

included Escherichia coli ATCC 35218, Pseudomonas aeruginosa, Staphylococcus aureus ATCC

25923, Bacilus subtilis, Streptococcus mutans ATCC 25175 and Salmonella enteric. The result showed

that two compounds have been isolated from E. elatior fruit, namely vanilic acid (1) and p-

hydroxybenzoic acid (2). The compounds and crude extracts were most active against S. mutans. The

data is a reference where the methanol extracts of E.elatior fruits can be developed into a mouthwash or

toothpaste.

1 INTRODUCTION

Zingiberaceae is one of the common plants in

Indonesia used as traditional medicines (Hartati et

al, 2014). The genus Etlingera belongs to the

Zingiberaceae family and contains approximately

150–200 species of worldwide distribution. Of

these, much species of this genus have been

recorded in Indonesia, including 48 species from

Sulawesi and 6 species from Java (Poulsen, 2012).

Species of this genus have been used in medicinal

folklore to treat various oilments, and the presence

of the volatile and non-volatile entities in these

species has gained research interests among

scientists.

Previous studies revealed the presence of

phenylpropanoids, flavonoids, and phytosterols in

the species of Etlingera. The leaves of E. elatior

produced quinic acid-containing cinnamic acid

derivatives, including 3-O-caffeoylquinic acid, 5-O-

caffeoylquinic acid (chlorogenic acid), and 5-O-

caffeoylquinic acid methyl ester (Chan et al, 2009a).

In addition, its leaves also contained kaempferol-3-

glucuronide, quercetin-3-glucuronide, quercetin-3-

glucoside, and quercetin-3-rhamnoside (Williams et

al, 1997). Moreover, leaves and rhizomes of E.

brevilabrum and E. sphaerochepala var. grandiflora

produced β-sitosterol and stigmasterol (Yahya et al,

2011; Mahdavi, 2014). The latter species also

yielded a simple phenolic paeonol (Mahdavi, 2014).

The stems of E. calophrys produced yakuchinone A,

p-hydroxybenzoic acid and stigmasterol (Sahidin et

al., 2018).

Different parts of Etlingera species also have proven

to have promising biological activities. Leaves and

stems of E. brevilabrum exhibited anticholesterol

activity (Mahdavi, 2014), while the leaves and

rhizomes of E. elatior performed antioxidant,

antibacterial, and tyrosinase inhibitory activities

Sahidin, I., Salsabila, S., Wahyuni, W., Malaka, M., Imran, . and Manggau, M.

Antibacterial Activity of Methanol Extracts and Compounds of Wualae (Etlingera elatior) Fruits from Southeast Sulawesi-Indonesia.

DOI: 10.5220/0008358901510154

In Proceedings of BROMO Conference (BROMO 2018), pages 151-154

ISBN: 978-989-758-347-6

151

(Williams et al, 1997; Ficker et al, 2003; Chan et al,

2008; Lachumy et al, 2010; Wijekoon et al, 2011;

Chan et al, 2009b, Chan et al, 2007). Antibacterial

and antioxidant activities was also exhibited by the

leaves extract of E. fulgens (Ficker et al, 2003).

Furthermore, antioxidant was also showed by

methanol extract of E. calophrys stems (Sahidin et

al., 2018). Other studies revealed the potency of E.

littoralis rhizomes and E. maingayi leaves as

antibacterial agents (Chiang et al, 2010). Moreover,

essential oil of Etlingera fenzlii (Kurz) K. Schaum

was safe for repellent source (Sudhakaran et al,

2016).

According to the above information, the chemistry

and pharmacology aspects of E. elatior fruits have

not been reported. Hence, the present work will

facilitate and report the isolation and identification

of chemical compounds from the methanol extract of

E. elatior fruits, as well as their antibacterial

activity.

2 MATERIALS AND METHODS

2.1 General Procedures

Instruments were used Cary Varian 100 Conc UV

spectrophotometer, PerkinElmer Spectrum One FT-

IR spectrophotometer, and JEOL ECP 500 NMR

spectrometer (500 MHz for

H and 125 MHz for

C). Chromatography techniques were performed

using Kieselgel 60 F

254

0,25 mm, silica gel 60 GF

254

and silica 60 G (Merck, Darmstadt, Germany). TLC

plates were derivatised using a cerium sulphate

reagent (Merck, Darmstadt, Germany). DPPH (2,2-

diphenyl-1-picrylhydrazyl) was purchased from

Merck (Darmstadt, Germany).

2.2 Sample

Fruits of Etlingera elatior were collected from the

Wolasi Forest, South Konawe, South East Sulawesi,

in November 2016 with No of Specimen EST02.

The plant specimen was identified and stored in the

Herbarium Bogoriense, Indonesia.

2.3 Extraction and Isolation

The dried powdered fruits of E. elatior (2.1 kg) was

macerated with methanol (MeOH, 3 x 5.0 L, 24 h

each time) at room temperature and yielded a dried

methanol extract as dark green gum (80 g). This

extract was further fractionated using a silica gel

VLC (10 x 5 cm, 150 g), eluted with n-hexane–ethyl

acetate (from 9:1 to 0:10) followed by pure MeOH,

and gave 5 main fractions (F1-F5) with weight of

1.3, 4.1, 7.3, 6.2, and 29.6 g, respectively. Main

fraction F3 was re-fractionated using a silica gel

VLC (10 x 5 cm, 150 g) and gradiently eluted with

n-hexane–ethyl acetate (from 7:3 to 0:10) and

MeOH as mobile phases, to yield subfractions F31

(0.1 g), F32 (0.7 g), F33 (0.7 g), and F34 (4.4 g).

Subfraction F32 was chromatographed using a

silica gel RC with chloroform–MeOH (95:5) and

pure MeOH as mobile phases, to produce pure

compound 1 (0.03 g). Furthermore, subfraction F33

was further purified using the same method as

compound 1 purification to get compound 2 (0.08 g).

2.4 Antibacterial Activity

The antibacterial assay was determined against

Bacillus subtilis FNCC 0060, Escherichia coli

ATCC 35218, Pseudomonas aeruginosa ATCC

27853, Salmonella enterica ATCC 14028,

Staphylococcus aureus ATCC 25923, and

Streptococcus mutans ATCC 25175. The

antibacterial test was conducted by the agar dilution

method using the general procedure outlined by

Thakurta (Sahidin et al, 2017). The cultural

concentration of bacteria was (B. subtilis = 2.0 x 10

cfu/mL, E. coli= 4.2 x 10

cfu/mL, P. aeruginosa =

1.2 x 10

cfu/mL, S. enterica = 2.0 x 10

cfu/mL, S.

aureus= 3.2 x 10

cfu/mL and S. mutans= 1.2 x 10

cfu/mL).

3 RESULTS AND DISCUSSION

3.1 Physicochemical Property and

Spectroscopic Data of the Isolated

Compounds from E. elatior Fruits

Two compounds (1–2) were successfully isolated

and identified from the methanol extract of E. elatior

fruits. Structures of these compounds were

determined based on their physicochemical property

and spectroscopic spectra of IR and NMR. These

data were also compared with the same data reported

in the previous studies.

Vanilic Acid (1); a white powder. Spectra of

NMR (500 MHz, CDCl

) δ

(ppm): 10.87 (br, s),

8.40 (1H, s), 7.61 (1H, dd, 8.4, 1.9), 7.58 (1H, d,

1.,7), 6.93 (1H, d, 8.2), and 3.92 (3H, s). Spectra of

C NMR (125 MHz, CDCl

) δ

(ppm): 166.6 (C-7),

BROMO 2018 - Bromo Conference, Symposium on Natural Products and Biodiversity

152

151.2 (C-4), 147.2 (C-3), 124.0 (C-6), 122.0 (C-1),

112.6 (C-5) and 55.4 (C-8).

p-Hydroxybenzoic acid (2); white amorphous

powder. Spectra of

H NMR (500 MHz, CDCl

) δ

(ppm): 9.43 (1H, s), 7.91 (2H, d, J = 8.6 Hz, H-2/H-

6), 6.92 (2H, d, J = 8.4 Hz, H-3/H-5). Spectra of

NMR (125 MHz, CDCl

) δ

(ppm): 166.7 (C-7),

161.8 (C-4), 131.8 (C-2/C-6), 121.8 (C-1), 115.1 (C-

3/C-5).

3.2 Antibacterial Activities Data

Table 1: Antibacterial activities of methanol extracts and the isolated compounds

Inhibition Zone (mm ± SD), [sample]= 100 μg/mL

Methanol

extracts

Vanilic Acid p-

Hydrox

ybenzoi

c aci

Chloramphenicol

. subtilis 0.30±0.11 0.00±0.00 0.17±0.10 15.30±0.80

E. coli 0.25±0.18 0.38±021 0.83±0.15 10.60±0.65

P. aeruginosa 0.30±0.05 0.80±0.14 0.17±0.12 9.71±0.90

S. enterica 0.30±0.18 3.80 ±0.30 0.63±0.20 12.70±0.75

. aureus 0.25±0.20 0.00±0.00 0.50±0.10 6.25±0.55

S. mutans 0.60±0.15 3.46±0.25 0.67±0.16 15.60±0.70

3.3 DISCUSSION

Vanilic acid (1) is firstly reported from Etlingera

plants. Meanwhile, p-hydroxybenzoic acid (2) has

been isolated from stems of E. callophrys (Sahidin

et al., 2018).

Those compounds were isolated from E. elatior

fruits are known compounds, so the structures are

determined by comparing the spectroscopic data of

isolated compounds with similar data from

references. For example, isolate 1, the spectrum data

H NMR and

C NMR has a high similarity

parameter with vanilic acid (1*) (Sheng et al, 2014).

It can be concluded that compound 1 is vanilic acid,

as displayed in Table 2.

Tabel 2: Spectra of

H and 13C NMR of isolate 1 and

vanilic acid

of C

Isolate 1

Vanilic Acid

(

Shen

et al., 2014

)

(∑H, m, J

in Hz

)

(∑H, m,

in Hz

)

C1 122.0 - 122.9 -

C2 115.6

7.58 (1H, d,

1,7

)

115.5

7.56 (1H, d,

1.7

)

C3 151.2 - 152.0 -

C4 147.2 8.4

(

1H,

)

148.0 -

C5 112.6

6.93 (1H, d,

8,2

)

113.4

6.91 (1H, d,

8.2

)

C6 124.0

7.61 (1H,

dd, 8,4, 1,9)

124.0

7.59 (1H,

dd, 8.2,

1.77)

C7 166.6 10.87

(

br,

)

167.5 -

C8 55.4 3.92 (3H,

) 56.3 3.82 (3H,

)

*Measured in acetone-d

(

H, 400 MHz;

C NMR

100 MHz)

In the same way as structure determination of

vanilic acid, the compound 2 is p-hydroxybenzoic

acid (Sahidin et al., 2018).

Based on biological activity data in the Table 1, The

activities of all samples both crude extracts and

isolated compounds are lower than chloramphenicol

(positive control) against some tested bacteria.

Methanol extract of E. elatior fruit at concentration

of 100 μg / mL (100 ppm) showed an interesting

Antibacterial Activity of Methanol Extracts and Compounds of Wualae (Etlingera elatior) Fruits from Southeast Sulawesi-Indonesia

153

antibacterial activity especially in inhibiting the

growth of S. mutans and S. enterica. This is

supported by the activity of compound successfully

isolated from the fruit of E. elatior which is vanilic

acid that has inhibition zone (mm) against S. mutans

and S. enterica are 3.46±0.25 and 3.80±0.30,

respectively. The data are references where the

methanol extracts of E. elatior fruits can be

developed into a mouthwash or toothpaste and anti-

salmonellosis diseases herbals.

4 CONCLUSION

Vanilic acid and p-hydroxybenzoic acid have been

isolated and identified from the methanol extract of

E. elatior fruits.. Of these, vanilic acid is firstly

isolated from the genus Etlingera. On biological

activities, potency of the crude methanol extracts as

an antibacterial agent was especially toward S.

mutans and S. enterica supported by the activity of

vanilic acid.

ACKNOWLEDGEMENTS

We would like to thank to Ministry of Research,

Science, Technology and Higher Education of

Republic of Indonesia for a research grant scheme

‘Hibah Penelitian Kompetensi” 2018 for the

financial support.

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