Bangladesh Journal of 
Pharmacology 

 Volume: 16; Number 4; Year 2021 
 

Cite this article as: Dat TTH, Oanh PTT. Pharmacological activities and volatile organic compounds from the 
endophytic bacteria from the leaves of Rhizophora apiculata. Bangladesh J Pharmacol. 2021; 16: 129-33. 

 



 

Pharmacological activities and volatile 
organic compounds from the endophytic 
bacteria from the leaves of Rhizophora 
apiculata    

 

Sir, 

The endophytic microorganisms with mangrove plants 
have been reported to be a prolific source of secondary 
metabolites with promising pharmacological proper-
ties. Of these, many compounds exhibit potent biologi-
cal activities and may be considered as lead compounds 
for further drug developments (Ancheeva et al. 2018). 
Rhizophora apiculata, a true mangrove plant, is widely 
distributed in mangrove forests in Vietnam. Phytoche-
mical and biological investigations on the endophytic 
microorganisms from R. apiculata reveal that they can 
produce secondary metabolites with valuable biological 
properties such as antibacterial, antifungal, antiviral, 
anti-cancer activities (Chaeprasert et al., 2010; Klaiklay 
et al., 2012; Fan et al., 2013; Zhou et al., 2019). Hence, 
the current study focused on the evaluation of pharma-
cological activities and volatile organic compounds of 
ethyl  acetate  extracts  from the  endophytic  bacteria 
isolated from the leaves of R. apiculata. 

Fresh leaves of R. apiculata were collected from Phu Loc 
District, Thua Thien Hue Province, Vietnam. The fresh 
leaves were surface-sterilized according to the protocol 
described elsewhere (Dat and Oanh, 2021). The surface-
sterilized leaves were crushed in sterile distilled water, 
and then 100 µL sample solution was spread on nutrie-
ent agar (NA, Himedia, India). The plates were incuba-
ted at 30ºC for 3-5 days. Colonies with different mor-
phological characteristics were identified by 16S rRNA 
gene sequences (Dat et al., 2021). 

From fresh leaves of R. apiculata, 10 strains with differ-
ent morphological features were selected for identifica-
tion using the 16S rRNA gene sequences. The molecular 
identification showed that the isolated strains belonged 
to 5 genera, i.e., Bacillus, Pseudomonas, Rossellomorea, 
Vibrio, and Staphylococcus (Supplementary file: Table S1; 
Figure S1). Interestingly, the majority of the isolated 
bacteria belonged to the genus Bacillus. 

The endophytic bacteria were used for producing ethyl 
acetate  extracts  and  evaluating  their  biological 
activities.  The extracts of the bacterial  strains were 

obtained by centrifuging the culture solutions at 10,000 
rpm for 10 min. Subsequently, the cell-free supernatants 
were  extracted  with  ethyl  acetate  (3  times)  and 
evaporated under reduced pressure to obtain the crude 
extracts. 

Biological properties of the bacterial extracts were 
evaluated. Antimicrobial activity of the extracts was 
determined by the microdilution (Dat et al., 2021), 
antioxidant and α-amylase and α-glucosidase inhibitory 
activities were determined using the protocol described 
elsewhere (Dat and Oanh, 2021), and xanthine oxidase 
inhibitory activity was determined using the protocol 
described elsewhere (Nguyen et al., 2004).  

Obtained results showed that 6 extracts exhibited anti-
microbial activity against at least one reference micro-
organism  with  minimum  inhibition  concentrations 
(MICs) from 16 to 256 µg/mL (Table I). Among them, 4 
extracts  exhibited  antimicrobial  activity  against  S. 
aureus, 3 extracts against E. faecalis, 4 extracts against E. 
coli,  2 extracts against P. aegurinosa,  and 3 extracts 
against C. albicans. Several extracts showed antimicro-
bial activity against multiple reference microorganisms. 
For example, 4 extracts exhibited antimicrobial activity 
against 3 reference microorganisms and two extracts 
against 2 reference microorganisms. Notably, several 
extracts showed significant antimicrobial activity with 
MIC values of 32-64 µg/mL. 

Antioxidant assays showed that 4 extracts exhibited 
DPPH radical scavenging activity with IC50 values from 
33.9 ± 0.7 to 62.2 ± 2.1 µg/mL and 5 extracts exhibited 
ABTS radical scavenging activity with IC50 values from 
53.4 ± 2.9 to 79.8 ± 5.4 µg/mL. Interestingly, two 
extracts, i.e., RAL_NA_4 and RAL_NA_8, exhibited 
both DPPH and ABTS radical scavenging activities. 

α-Amylase and α-glucosidase assays showed that 4 
extracts exhibited α-amylase inhibitory activity with 
IC50 values from 63.2 ± 1.4 to 130.1 ± 6.8 µg/mL and two 
extracts exhibited α-glucosidase inhibitory activity with 
IC50 values from 57.8 ± 1.3 to 175.0 ± 6.8 µg/mL. How-
ever, only one extract (RAL_NA_2) exhibited both α-
amylase and α-glucosidase inhibitory activities. Regar-
ding the xanthine oxidase, 4 extracts exhibited xanthine 
oxidase inhibitory activity with IC50 values from 56.9 ± 
2.8 to 77.2 ± 3.7 µg/mL (Table I). 

The volatile organic compounds in the extract of the 
most promising bioactive strain Bacillus sp. RAL_NA_8 

A Journal of the Bangladesh Pharmacological Society (BDPS) Bangladesh J Pharmacol 2021; 16: 129-133 
Journal homepage: www.banglajol.info; www.bdpsjournal.org 
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ISSN: 1991-0088; DOI: 10.3329/bjp.v16i4.56682 

Letter to the Editor 

This work is licensed under a Creative Commons Attribution 4.0 International License. You are free to copy, distribute and perform the work. 
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were investigated by the GC–MS analysis using the 
Agilent 7890B gas chromatograph-assisted Agilent 
5977A mass detector (Agilent Technologies, USA). The 
compounds were identified by comparing the spectra 
with a stored MS library (W8N08 and NIST08) and the 
relative percent of individual components was calcula-
ted based on GC peak areas. The GC-MS analysis iden-
tified 11 volatile compounds in the extract, including 
major compounds 2,5-furandione, dihydro (62.8%), 
[(3R,4s,5S)-4-nitrothian-3,5-diyl]-diacetate (11.2%), 1,3-
dioxolane, 2,2,4,5-tetramethyl-, trans- (8.4%), 2-(methyl-
D3)-cycloheptanone (4.0%), and hyacinthin (3.2%) 
(Table II; Supplementary file: Figure S2).  

 The results in the present study are consistent with 
previous investigations of bioactive natural products 
from endophytic microorganisms with the mangrove 
plant R. apiculata (Chaeprasert et al., 2010; Klaiklay et 

al., 2012; Fan et al., 2013; Zhou et al., 2019). For 
example, Klaiklay et al. (2012) isolated two cytotoxic and 
antibacterial compounds from the endophytic fungus 

Phomopsis sp. PSU-MA214. The isolated compounds 

exhibited cytotoxicity against the cancer cell line MCF-7 and 

KB with IC50 of 27-43 μg/mL and antibacterial activity 
against S. aureus and methicillin-resistant S. aureus with 
MICs of 64-128 μg/mL. Zhou et al. (2019) isolated three 
antimicrobial compounds, fusolanone A-B and fusaric 
acid, from the endophytic fungus Fusarium solani 
HDN15-410. These compounds exhibited good 
antimicrobial activity against Pseudomonas aeruginosa, 
Monilia albican, Bacillus subtilis, and Vibrio parahaemoly-
ticus with MICs of 6.25–50 μg/mL. 

In conclusion, the endophytic bacteria from the leaves 
of R. apiculata may be a potential source of pharmaco-
logical secondary metabolites. 

130 Bangladesh J Pharmacol 2021; 16: 129-133         

Table I 

Biological activity of the bacterial extracts 

Strain ID 

Antimicrobial activity (MIC, μg/mL) 

S. aureus 
ATCC 25923 

E. faecalis 
ATCC 29212 

E. coli 
ATCC 25922 

P. aegurinosa 
ATCC 27853 

C. albicans 
ATCC 10231 

RAL_NA_1 - - - - - 

RAL_NA_2 - - 64 256 - 

RAL_NA_3 - - - - - 

RAL_NA_4 128 - 256 - 64 

RAL_NA_5 32 64 - - 128 

RAL_NA_6 - - 32 - 256 

RAL_NA_7 - - - - - 

RAL_NA_8 64 32 - 128 - 

RAL_NA_9 256 256 128 - - 

RAL_NA_10 - - - - - 

Ciprofloxacin 1 2 0.5 0.5 - 

Fluconazole         2 

Strain ID 

Antioxidant activity (IC50, μg/mL) Enzyme inhibitory activity (IC50, μg/mL) 

DPPH radical scav-
enging 

ABTS radical scav-
enging 

α-Amylase α-Glucosidase Xanthine oxidase 

RAL_NA_1 >100 >100 >200 >200 77.17 ± 3.71 

RAL_NA_2 >100 >100 82.6 ± 3.4 57.8 ± 1.3 >100 

RAL_NA_3 >100 79.1 ± 2.9 76.50 ± 2.15 >200 63.5 ± 1.4 

RAL_NA_4 62.5 ± 2.1 54.8 ± 1.5 >200 >200 >100 

RAL_NA_5 43.8 ± 2.1 >100 63.2 ± 1.4 >200 >100 

RAL_NA_6 >100 70.1 ± 2.0 >200 >200 71.5 ± 2.1 

RAL_NA_7 >100 >100 >200 175.0 ± 6.8 >100 

RAL_NA_8 57.0 ± 0.1 53.4 ± 2.9 130.1 ± 6.8 >200 >100 

RAL_NA_9 >100 79.8 ± 5.4 >200 >200 56.9 ± 2.8 

RAL_NA_10 33.9 ± 0.7 >100 >200 >200 >100 

Ascorbic acid 28.0 ± 0.6 25.0 ± 0.6       

Acarbose - - 82.4 ± 1.0 205.4 ± 6.3 - 

Allopurinol - - - - 4.1 ± 0.3 

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This  work  was  supported  by  Vietnam Academy of Science 
and Technology (Grant No.: ĐL0000.02/19-20). 

 

Ton That Huu Dat and Phung Thi Thuy Oanh 

Mientrung Institute for Scientific Research, Vietnam National Museum of 
Nature, Vietnam Academy of Science and Technology, 321 Huynh Thuc 
Khang, Hue city, Thua Thien Hue, Vietnam. 

Corresponding author: 

Email: tthdat@vnmn.vast.vn  

References 

Ancheeva E, Daletos G, Proksch P. Lead compounds from 
mangrove-associated microorganisms. Marine Drugs. 2018; 
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Chaeprasert S, Piapukiew J, Whalley AJ, Sihanonth P. Endo-
phytic fungi from mangrove plant species of Thailand: Their 
antimicrobial and anticancer potentials. Bot Mar. 2010; 53: 
555-64.  

Dat TTH, Cuc NT, Cuong PV, Smidt H, Sipkema D. Diversity 
and antimicrobial activity of Vietnamese sponge-associated 
bacteria. Marine Drugs. 2021; 19: 353.  

Dat TTH, Oanh PTT. In vitro antioxidant, α-amylase and α-
gluco-sidase inhibitory activities of endophytic bacteria 
from the roots of the mangrove plant Rhizophora stylosa 
Griffith. Acad J Biol. 2021; 43: 125-35.   

Fan Y, Wang Y, Liu P, Fu P, Zhu T, Wang W, Zhu W. Indole-
diterpenoids with anti-H1N1 activity from the aciduric 
fungus Penicillium camemberti OUCMDZ-1492. J Nat Prod. 
2013; 76: 1328-36.  

Klaiklay S, Rukachaisirikul V, Phongpaichit S, Pakawatchai C, 
Saithong S, Buatong J, Preedanon S, Sakayaroj J. Anthraqui-
none derivatives from the mangrove-derived fungus 
Phomopsis sp. PSU-MA214. Phytochem Lett. 2012; 5: 738-42.   

Nguyen MT, Awale S, Tezuka Y, Le Tran Q, Watanabe H, 
Kadota S. Xanthine oxidase inhibitory activity of Vietna-
mese medicinal plants. Biol Pharm Bull. 2004; 27: 1414-21.   

Zhou G, Qiao L, Zhang X, Sun C, Che Q, Zhang G, Zhu T, Gu 
Q, Li D. Fusaricates HK and fusolanones AB from a man-
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Table II 

Volatile organic compounds in the extract of    Ba-
cillus sp. 

Compounds RT/min Quantity 
(%) 

Diacetone alcohol 5.671 1.23 

2,2,3,4-Tetramethyl-hex-5-en-3-ol 5.939 1.97 

2-(Methyl-D3)-cycloheptanone 6.271 4.03 

4,6,8-Trimethyl-,9-undecan-5-ol 6.441 1.70 

Oxirane, 2,2-dimethyl-3-propyl 8.182 2.74 

Hexyl ethyl carbinol 8.496 1.24 

1,3-Dioxolane, 2,2,4,5-tetramethyl-, 
trans- 

8.693 8.35 

[(3R,4s,5S)-4-nitrothian-3,5-diyl]-
diacetate 

9.042 11.21 

4-Heptanol, 4-ethyl-2,6-dimethyl 9.473 1.48 

2,5-Furandione, dihydro 10.181 62.83 

Hyacinthin 10.515 3.21 

Table SI 

The closest 16S rRNA sequences of the isolated strains obtained in NCBI GenBank 

Strain ID Sequence length (nt) NBCI closest homologs Identity (%) 

RAL_NA_1 1419 Staphylococcus argenteus BG-V-5, OK326042 99.93 

RAL_NA_2 1409 Bacillus cereus SW9SE, MN068934 100 

RAL_NA_3 1379 Bacillus pseudomycoides KUBOTAB2, MK855402 99.86 

RAL_NA_4 1420 Bacillus subtilis, AB192294 100 

RAL_NA_5 1449 Bacillus infantis C4, MF993020 99.87 

RAL_NA_6 1398 Pseudomonas aeruginosa ATCC 27853, AF094719 99.93 

RAL_NA_7 1379 Vibrio cholerae TS5-B, LC487859 99.85 

RAL_NA_8 1400 Bacillus altitudinis 19RS3, MH883312 99.94 

RAL_NA_9 1449 Bacillus baekryungensis LS218, FJ937928 99.81 

RAL_NA_10 1440 Rossellomorea vietnamensis 151-6, CP047394 100 

Supplementary 

 Bangladesh J Pharmacol 2021; 16: 129-133        131 

mailto:tthdat@vnmn.vast.vn


 

Figure S1: Phylogeny of 16S rRNA gene sequences of the endophytic bacteria from the present study (bold letters) and from NCBI 
GenBank. Bootstrap support values of branches greater than 75% are given above the corresponding branches 

132 Bangladesh J Pharmacol 2021; 16: 129-133         



 

Figure S2: GC-MS chromatogram of the ethyl acetate extract of Bacillus sp. RAL_NA_8 

Time (min) 

 Bangladesh J Pharmacol 2021; 16: 129-133        133