Chemical Compounds from Fungus Syncephalastrum racemosum

Isolated as Endophytic from Ageratum conyzoides

Elfita

, Muharni

, Mardiyanto

, Fitrya

and Rismawati Simangunsong

Department of Chemistry, Faculty of Mathematics and Natural Sciences, University of Sriwijaya, Inderalaya,

Kabupaten Ogan Ilir 30662, Indonesia

Department of Pharmacy, Faculty of Mathematics and Natural Sciences, University of Sriwijaya, Inderalaya,

Kabupaten Ogan Ilir 30662, Indonesia

Keywords: Ageratum conyzoides, Endophytic Fungus, Syncephalastrum racemosum.

Abstract: Ageratum conyzoides known as bandotan is a plant widely grown in Indonesia. This plant is used for the

treatment of various diseases such as antibacterial, anti-diabetic, anti-inflammatory, antioxidant, and

analgesic. The active compounds contained in this plant include alkaloids, flavonoids, tannins, glycosides,

minerals and other compounds. Plants that have ethno medicine history are promising candidates to obtain

bioactive compounds from their endophytic fungi. In the present study, chemical compounds were isolated

from endophytic fungus Syncephalastrum racemosum from the stem of Ageratum conyzoides with the

chromatography method. The structures of the compounds were determined by spectroscopy analysis. The

compounds are aromatic group.

1 INTRODUCTION

Ageratum conyzoides is known as bandotan. In some

countries, bandotan is considered a weed plant and

its growth is very fast. This plant comes from

tropical America, especially Brazil. Most of the A.

conyzoides plants are found in Mexico, Central

America, the Caribbean Islands, and Florida. But

now bandotan is also found in several sub-tropical

and tropical countries, including in Indonesia.

Bandotan plants are now widespread in various parts

of Indonesia. Ageratum conyzoides often grow in

yards, roadside, fields, dry rice fields, river banks,

and areas with a lot of shrubs. This plant has a long

history in its use for traditional medicine in several

countries. This plant has medicinal bioactive

properties. Therefore bandotan plants can be

classified as herbal plants (Soerjani et al., 1987;

Darma, 1987; Singh et al., 2013; Odeleye et al.,

2014; Janarthanan et al., 2016)

In general A. conyzoides contains a variety of

bioactive compounds including flavonoids,

alkaloids, coumarins, essential oils, tannins,

chromene, benzofuran and terpenoids. All parts of

this plant have the ability to be anti-inflammatory

and anti-allergic. In addition, antidiareal,

nematoside, anticoagulant, smooth muscle relaxant,

hemostatic, analgesic, antifungal, antibacterial, and

hypothermic factors are also reported (Kamboj and

Saluja, 2008; Ndip et al., 2009; Awad et al., 2013;

Bahtiar et al., 2017)

In Bogor, A. conyzoides is widely known as a

wound medicine. According to Heyne, these plant

leaves are squeezed, mixed with lime, applied to

fresh wounds. Decoction of leaves is also used to

treat chest pain, while extracting the leaves for eye

drops. Mashed roots are applied to the body to treat

fever, the extract can be drunk. bandotan also to treat

stomach ache and to cure broken bones (Heyne,

1987; Darma, 1987).

Endophytic fungi are microorganisms that live to

form colonies in plant tissues without endangering

their host plants. Each high-level plant contains

several endophytic microbes which produce

secondary metabolites as a result of coevolution or

genetic transfer (genetic recombination) from the

host plant to endophytic fungi. The ability of

endophytic fungi to produce phytochemical

compounds that are also produced by their host

plants may be related to the presence of genetic

recombination of endophytic fungi with hosts during

the time of their evolution (Elfita et al, 2013; Sandhu

et al., 2014; Golinska et al., 2015).

136

Elﬁta, ., Muharni, ., Mardiyanto, ., Fitrya, . and Simangunsong, R.

Chemical Compounds from Fungus Syncephalastrum racemosum Isolated as Endophytic from Ageratum conyzoides.

DOI: 10.5220/0008858001360140

In Proceedings of the 1st International Conference on Chemical Science and Technology Innovation (ICOCSTI 2019), pages 136-140

ISBN: 978-989-758-415-2

Endophytic fungi are a source of genetic

diversity with various possible new species that have

not been described. Therefore, the need for natural

products for new antibiotics, chemotherapy and

agrochemicals that have high activity, low toxicity,

but do not disturb environmental ecology can be

expected to be obtained from this endophytic fungus

(Rajamanikyam et al., 20017; Kaur et al., 2018;

Santoyo et al., 2016; Elfita et al., 2015).

Previous studies have reported six endophytic

fungi isolated from the leaves and stems of

Ageratum conyzoides (Elfita et al., 2019). Screening

the antibacterial activity of ethyl acetate extract from

liquid culture showed that the Syncephalastrum

racemosum fungus had the highest activity. In this

paper, the chemical compounds contained in the

antibacterial active extract of the S. racemosum

fungus are reported.

2 MATERIALS AND METHODS

2.1 Chemicals

The materials used in this study include endophytic

fungi, Syncephalastrum racemosum which have

been previously isolated, Potato Dextrose Agar

(PDA), Potato Dextrose Broth (PDB), alcohol 70%,

KLT kiessel gel 60 F254 20 x 20 cm, silica gel G 60

70-230 mesh. Organic solvents such as n-hexane,

ethyl acetate and methanol.

2.2 Source of Endophytic Fungi

Syncephalastrum racemosum of Ageratum

conyzoides from stock fungus (Elfita et al., 2019).

The fungal was identified molecularly in the

biological research center-LIPI Cibinong. The

Ageratum conyzoides was collected in Juli 2018

from Indralaya, Ogan Ilir, South Sumatra.

2.3 Cultivation and Extraction of

Endophytic Fungi S. racemosum

The suspension of endophytic fungus (which was

previously isolated) was prepared by taking 6 ose of

endophytic fungi, then inoculated into 200 mL of

PDB (in 5x 1 liter bottles). They were incubated at

room temperature and static conditions for 6 weeks.

Furthermore, endophytic fungal cultures are

harvested by separating biomass. the liquid culture

was extracted using ethyl acetate in a separating

funnel. then the extract was evaporated using a

rotary evaporator to obtain concentrated ethyl

acetate extract (Marcellano et al., 2017; Elfita et al.,

2012).

2.4 Isolation of Secondary Metabolites

and Identification of Structures

The ethyl acetate concentrate extract of endophytic

fungi was separated by chromatography column.

The sample was prepared by preadsorption and put

into the column over silica gel 70-230 mesh.

separation was carried out using using eluents with

gradient system (n-hexane-EtOAc-methanol). The

eluates was collected with a vial (10 mL) and

analyzed by TLC under UV lamp. The eluate with

the same stain pattern was combined into one

fraction. The major and fluorinated stain fractions

were then separated and purified. Sub-fractions were

separated again using column chromatography over

silica gel (70-230 mesh) with gradient eluents.

Eluate is collected in a vial (5 mL volume) and

analyzed by TLC. Eluat with the same stain pattern

were combined into one fraction. The subfraction re-

purified column chromatography to obtain the pure

compounds. The compounds were analyzed for

their chemical structure using spectroscopic

methods.

3 RESULT AND DISCUSISON

3.1 Isolation of Chemical Compounds

The filtrate was evaporated by rotary evaporator to

give a EtOAc extract (4.1 g). The extract (4.1 g) was

separated over a silica gel column (70.230 mesh, 40

g) with gradient solvent system of n-

hexane/EtOAc/MeOH as the eluent to give five

fractions (A1–A5). Fraction A1 (2.34 g) was

subjected to column chromatography (CC) eluted

with n-hexane-EtOAc (10:0→0:10) as to give four

sub-fractions (A11–A14). Subfraction A12 (1.1 g)

was subjected to CC over a silica gel (70-230 mesh,

20 g) eluted with with n-hexane-EtOAc (9:1) to

give compound 1 (479 mg). Fraction A3 (709 mg)

was separated to CC eluted with n-hexane-EtOAc

(7:3→0:10) as to give five sub-fractions (A31–A35).

Subfraction A31 (91 mg) was subjected to CC over a

silica gel (70-230 mesh, 10 g) eluted with with n-

hexane-EtOAc (5:5) to give compound 2 (21 mg).

Subfraction A35 (56 mg) was subjected to CC eluted

with n-hexane-EtOAc (4:6) to give Compound 3 (11

mg, not identified).

Chemical Compounds from Fungus Syncephalastrum racemosum Isolated as Endophytic from Ageratum conyzoides

137

3.2 Identification of Chemical

Compounds

Compound 1 The UV spectrum of compound 1 in

methanol solvents (Figure 1) showed absorption at

max

224 and 274 nm. Addition of NaOH does not

cause a batochromic shift. This indicates the absence

of a free hydroxyl group on the aromatic ring

(Muharni et al., 2014).

Figure 1: The UV spectrum of compound 1.

The FTIR spectrum (Figure 2) showed the

presence of characteristic absorption bands at υ

3344.3 cm

-1

which is absorption for OH, while the

absorption of 2854.7-2958.8 cm

-1

is a typical for

aliphatic C-H. In addition there is absorption in the

area of 1728.22 cm

-1

which is absorption for C = O

bonds. The presence of aromatic C = C is

characterized by absorption at 1600.9 and 1462.04

-1

and typical absorption of C-O ester in the area

of 1274.9 cm

-1

Figure 2: The FTIR spectrum of compound 1.

Figures 3 showed the presence of 6 proton

signals. The signal at 

0,89 ppm (12H, m) for 4

methyl groups. Furthermore, the signal that

accumulates in the area around 

1,30-140 ppm (

18H, m) showed the presence of aliphatic CH

groups. In the

H-NMR spectrum also shows that the

signal in the area of 1.68 ppm (2H, m) is a signal for

two CH groups coupled by protons through three

bonds.

The signal at 

4,22 ppm (2H, m) showed the

presence of O-CH

group (2 CH

groups).

Furthermore, the signal in the area of 

7,52 and 

7,70 ppm (2H, dd J = 3.4 and 5.8 Hz) respectively

showed the presence of four aromatic protons

(coupled meta and ortho). Each signal represented

two protons. the compound 1 as an aromatic ring in

the form of symmetrical disubstitution (Habib and

Karim, 2009).

Figure 3: The

H-NMR spectrum of compound 1 (500

MHz

H- in CDCL

The

C-NMR spectrum (Figure 4) showed the

presence of 12 signals consisting of 8 C sp3 signals

that appear below 100 ppm ( 

11.1; 14.2;

23.1;

23.9;

29.1;

30.5;

38.9; and 68.3 ppm ) and 4 other

signals that appear above 100 ppm ( 

128.9;

131.0;

132.6; and

167.8 ppm). are signals for C sp

Figure 4: The

C-NMR spectrum of compound 1 (125

MHz

C- in CDCL

The

H-NMR spectrum of compound 2 (Figure 5)

showed a group of protons similar to compound 1. It

appears that compound 2 also has signals in the

regions 

7,53 dan 

7,71 ppm (2H, and J = 3.3

and 5.7Hz) respectively showed the presence of four

aromatic protons (coupled meta and ortho). Each

signal represented two protons. The compound 2 as

an aromatic ring in the form of symmetrical

disubstitution. The next similar signal, at 

4,22

ppm (2H, m) for O-CH

group (2 CH

groups) and at



0.5-2.00 ppm is a long chain of aliphatic protons.

The difference is the appearance of a proton signal at

5007501000125015001750200025003000350040004500

1/cm

-0

3433.29

2958.80

2926.01

2854.65

1728.22

1600.92

1462.04

1381.03

1274.95

1122.57

1072.42

958.62

742.59

Swl2

ICOCSTI 2019 - International Conference on Chemical Science and Technology Innovation

138



3.66 ppm which is a methoxyl proton (Habib and

Karim, 2009; Muharni et al., 2014).

Figure 5: The

H-NMR spectrum of compound 2 (500

MHz

H- in CDCL

4 CONCLUSIONS

Chemical compounds isolated from the ethyl acetate

extract of liquid cultured of endophytic fungi

Syncephalastrum racemosum were identified as

phthalate derivatives.

ACKNOWLEDGMENTS

The authors are thankful to ministry Ristekdikti

Republic of Indonesia, which provides funding with

the research grant Penelitian Dasar Unggulan

Perguruan Tinggi (PDUPT) 2019.

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