Identification of Bioactive Compounds of Ficus septica
Leaf Extract has Potential as Botanical Pesticides to
Control Anthracnose Disease on Chili Pepper
By
Sang Ketut Sudirga and I Ketut Ginantra
ISSN 2319-3077 Online/Electronic
ISSN 0970-4973 Print
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J. Biol. Chem. Research
Volume 34 (1) 2017 Pages No. 150-159
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An International Peer Reviewed / Referred Journal of Life Sciences and Chemistry
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Identification of…………………..……on Chili Pepper Sudirga and Ginantra, 2017
J. Biol. Chem. Research. Vol. 34, No. 1: 150-159, 2017
(An International Peer Reviewed / Refereed Journal of Life Sciences and
Chemistry)
Ms 34/1/70/2017
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ISSN 0970-4973 (Print)
ISSN 2319-3077 (Online/Electronic)
Sang Ketut Sudirga
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[email protected] RESEARCH PAPER
Received: 20/02/2017 Revised: 18/03/2017 Accepted: //2017
Identification of Bioactive Compounds of Ficus septica
Leaf Extract has Potential as Botanical Pesticides to
Control Anthracnose Disease on Chili Pepper
Sang Ketut Sudirga and I Ketut Ginantra
Department of Biology, Faculty of Mathematics and Natural Sciences, Udayana University,
Kampus Bukit Jimbaran Bali Indonesia
ABSTRACT
Based on the preliminary study as many as 20 species of plants extract found that the
crude extract of the leaves of Ficus septica able to inhibit the growth of Colletotricum
acutatum, its fungus the cause of anthracnose disease on chili pepper. Base on in vitro
test on PDA with inhibition zone diameter of 30 mm, but it is not certain bioactive
compounds. For this problem, the aim is research is conducted to determine the content of
bioactive compounds potentially as botanical pesticides. The method used is the method
of column chromatography and thin layer, and GCMS. The study states that the extracts of
Ficus septica containing 4 secondary metabolites are terpenoids, alkaloids, flavonoids, and
phenols. Result analysis using GCMS there are 14 active compound namely dl-
glyceraldehyde dimer, 2,3,5 trimethyl heptane, Sulfurous acid cyclohexylmethylhexadecyl
ester, guanosine, D-Allose, dodecanoic acid methyl ester, 1,2-Benzenedicar boxylic acid
diethyl ester, 3-Deoxy-d-mannonic acid, cyclohexane tetraethyl 1,2,3,4, (Z) - 9-Tricosene,
hexadecanoic acid methyl ester, octadecamethylcyclononasi-loxane, 1-Heptacosanol and
1,2-Benzene dicarboxylic acid mono (2-etilhexyl) ester. Based on the existing references, of
14 compounds and 8 of them have been known as antifungal compounds. Those are 2,3,5
trimethyl heptane, hexadecylcyclohexylmethyl Sulfurous acid ester, dodecanoic acid
methyl ester, 3- deoxy-d-mannonic acid, hexadecanoic acid methyl ester,
octadecamethylcyclononasiloxane, 1-Heptacosanol and 1,2-Benzenedicar boxylic acid
mono (2-etilhexyl) ester.
Keywords: Antifungal, Ficus septica, Colletotrichum acutatum, Anthracnose Disease and
Botanical Pesticides.
Identification of…………………..……on Chili Pepper Sudirga and Ginantra, 2017
INTRODUCTION
Anthracnose disease in chili pepper is the most common disease and almost always occurs
in every area of chili plants (Figure 1B). According to Suryaningsih et al. (1996), the most
common cause of anthracnose in chili plants in Indonesia is Colletotrichum capsici and
Colletotrichum gloeosporioides. According to (Sudiarta and Sumiartha, 2012; Sudirga, S.K.
2016) anthracnose disease on chili plants in Bali is mostly caused by Colletotrichum
acutatum. Anthracnose disease can damage the aesthetic value of the chili and lowering the
yield to 50% or more (Semangun, 2007). Anthracnose disease control today still relies on
the use of synthetic fungicides. The use of synthetic fungicides continuously may lead to the
emergence of pathogen resistance, pollute the environment and is harmful to consumers.
Based on this, it is necessary to look for an alternative control of anthracnose disease on
chili plants by utilizing the potential crop as botanical fungicide that is not dangerous for
consumers and the environment. According Sudirga et al. (2014) in the preliminary study as
many as 20 species of plants have been tested in terms of antifungal activity against
Colletotrichum acutatum the cause of anthracnose disease on chili pepper, and found six
kinds of plants that can inhibit the growth of C. acutatum. These 6 kinds of plants are Ficus
septica, Albizia saman, Piper nigrum, Piper crocatum, Piper retrofectum and Thitonia
difersifolia. Among the six species, F. septica leaf extract has the highest inhibitory activity
with inhibition zone of 30 mm. Some plant species are reported to have antifungal activity
such as Ageratum conyzoides has antifungal activity against Penicillium italicum (blue mold)
the cause of fruit rot disease on Mandarin orange (Dixit et al., 1995); Origanum manjorona
has antifungal activity against Colletotrichum gloeosporioides the cause of anthracnose
disease on coffee (Silva et al., 2008); Albizia saman has antifungal activity against Fusarium
sp. the cause of wilt disease in chili plants (Suprapta and Khalimi, 2012). Awar-awar (Ficus
septica Burm.f.) is a wild plant and by the community it is only used as a traditional medicine
(Figure 1A). Vital et al. (2010) reported a crude extract of awar-awar leaves can inhibit the
growth of Staphylococcus aureus, Canida albicans and Escerechia coli with inhibition zones
respectively 14 mm, 18 mm and 13 mm. Suspected chemical compound contained in the
leaves, fruits and roots of awar-awar in the form of alkaloids, saponins, flavonoids, tannins
and polyphenols (de Padua et al., 1999).
Figure 1. A = Leaf of Ficus septica, B= Anthracnose disease on chili pepper.
(Source : private collection , 2014)
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Sukadana (2010) reported the extracts of the root bark of awar-awar (Ficus septica Burm.f.)
contains flavonoid compounds from the class of flavanones and these compounds can
inhibit the growth of bacteria Vibrio cholerae and Escherichea coli. Damu et al. (2005)
reported the extracts of stem awar-awar containing alkaloids compounds from the class of
alkaloids phenanthro-indolizidine consisting of ficuseptines BD (1-3), 10R, 13aR-tylophorine
N-oxide (4), 10R, 13aR-ylocrebrine N-oxide ( 5), 10S, 13aR-tylocrebrine N-oxide (6), 10S,
13aR-isotylocrebrine N-oxide (7), and 10S, 13aS-isotylocrebrine N-oxide (8). The alkaloid
classes of compounds are cytotoxic. According to Nugroho et al. (2011) results from the
fractionation of ethanol and hexane of awar -awar leaf extract has potential as an
anticancer compound. Besides, leaves and roots awar-awar contains saponins and
flavonoids, fruits contain alkaloids and tannins, and roots contain polyphenols (de Padua et
al., 1999).
Crude extract of leaves of Ficus septica able to inhibit the growth of Colletotrichum
acutatum fungus in vitro on PDA with inhibition zone diameter of 30 mm, but it is not
certain bioactive substances. For these conditions, this study is a follow-up study, conducted
to determine the content of bioactive substances potentially as botanical pesticides from
leaves extract of F. septica.
MATERIAL AND METHODS
Methods of Extraction
Extraction of awar-awar leaves was done by chopping the leaves, and then dried at room
temperature, and after the dry material was made into powder by means of a blender.
Awar-awar leaf powder (100 grams) was then macerated with 1000 ml of methanol PA (Pro
Analysis) for 72 hours at room temperature and dark place. The filtrate was obtained by
filtering and the residue obtained was then macerated again with 1000 ml of methanol as
much as two times. The filtrate obtained are combined and then evaporated using a vacuum
rotary evaporator (Iwaki, Japan) at 40°C, to obtain a crude extract that was used for further
testing.
Antifungal Activity Test
Antifungal activity test of crude extract of the leaves of awar-awar against Colletotrichum
acutatum was done in well diffusion method. According to Ardiansyah (2005), if the
diameter of inhibition zone is ≥ 20 mm the inhibitory activity is very strong; 10-20 mm the
inhibitory activity is strong; 5-10 mm the inhibitory activity is moderate; and ≤ 5 mm the
inhibitory activity is poor or weak.
Analysis of Phytochemicals
Phytochemical analysis was conducted to determine the compound of the active fraction
obtained by using reagents for specific classes of compounds. The compounds of the active
components tested included: terpenoids, alkaloids, flavonoids, phenols, saponins, and
tannins. Analysis was performed on fractions which showed the highest antifungal
properties (Harborne, 1989).
Separation and Purification of Active Extracts
The crude extract of awar-awar was partitioned with n-hexane and methanol to obtain the
extract phase of n-hexane and methanol phase. Furthermore, both the extracts were tested
for the antifungal activity.
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Identification of…………………..……on Chili Pepper Sudirga and Ginantra, 2017
The separation and purification was performed by column chromatography using silica gel
(60 from 0.063 to 0.200 mm) as stationary phase, while the mobile phase is a mixture of
various kinds of solvents which are based on differences in polarity. From the
chromatography column, it produced several fractions and each fraction was tested for
antifungal activity. Some active fractions were fractionated again using the same eluent as
the previous fractionation. Each fraction obtained in the second fractionation was tested for
antifungal activity and further active fractions were analyzed by KLT to determine the spot
patterns generated from each of the fractions. Fractions that produce the same spot pattern
were incorporated as a combined fraction of and tested for antifungal activity. The most
active fraction was then analyzed by GC-MS to know the types of chemical compounds
contained in this fraction.
RESULTS AND DISCUSSION
Inhibitory Activity of Partitioned Extract
Based on the results of partition using counter-current distribution method with two types
of solvents are hexan and methanol phase showed that the methanol extract could inhibit
the growth of Colletotrichum acutatum with the diameter of inhibition zone of 30 mm,
whereas hexane extract phase could not inhibit the growth of this fungus (Figure 2). These
results indicate that the active compounds in the leaf extract of awar-awar that are
antifungal against C. acutatum is in the phase of methanol and is polar.
A B
1 1
2 2
3
Figure 2. Photos of inhibition zone formed around the well diffusion filled with partitioned
leaf extract of awar-awar of methanol phase (A) and hexane phase (B). (1 = mycelium of
C. acutatum, 2 = well diffusion, and 3 = inhibition zone)
Gawade at al. (2014) reported the leaf extract of Aegle Marmelos (L). can inhibit the growth
of the fungus Colletotrichum acutatum with inhibition zone diameter of 22 mm. According
to Nogodula et al. (2012) crude extract of leaves of awar-awar is able to inhibit the growth
of mold Canida albicans inhibition zone with a diameter of 16.67 mm, but has not been any
report on the bioactive compound of leaf extract of awar-awar has potential as botanical
fungicides to control anthracnose disease cause of C. acutatum on chili pepper.
Inhibitory Activity of Fractionated Extract
Methanol phase fractionation by column chromatography produces 44 fractions. All
fractions tested for inhibitory activity against C. acutatum of on PDA media with well
diffusion method. Five active fractions were found to inhibit the growth of C. acutatum
namely fractions 40, 41, 42, 43, and 44 with the diameter of inhibition zone respectively 20
mm, 25 mm, 29 mm, 29 mm and 25 mm ( Figure 3).
J. Biol. Chem.
A Research 153 Vol. 34 (1): 150-159 (2017)
B C D E
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Figure 3. Inhibition zone of 5 active fractions of 44 fraction results of methanol phase
fractionation of leaf extract of awar-awar (A = fraction 40, B = fraction 41, C = fraction 42,
D = fraction 43 and E = fraction 44).
All five active fractions were combined into combined fractions and were fractionated again
by column chromatography using eluent same as the previous fractionation. Eighteen
fractions which showed strong antifungal activity against C. acutatum was analyzed using
thin layer chromatography (TLC) to determine the pattern of the spots in each fraction. The
results showed that of the 18 fractions were tested showed a pattern of spots and with
almost the same Rf value i.e. between 0.7 and 0.8 so it can be presumed that the active
compounds contained among the 18 active fraction possibilities belong to a group or class
of similar compounds. The size of the inhibition of a plant extract against fungus varies
greatly depending on the type and concentration of compounds (Suprapta, 2001). Castillo et
al. (2012) reported awar-awar contain active compounds antofine and ficuseptine. Antofine
compound has potential as an anticancer compound while the compounds ficuseptine
potential as antibacterial and antifungal compounds. Results fractionation of ethanol and
hexane awar- awar leaf extract has potential as an anticancer compound, in addition to
leaves, fruits and roots awar - awar contain alkaloids, saponins and flavonoids as a potential
antimicrobial compounds (Nugroho et al., 2011).
Phytochemicals of the Leaf Extract of Awar-Awar
Phytochemical test of the methanolic leaf extract of awar-awar showed that the leaf extract
of awar-awar containing compounds such as terpenoids, alkaloids, flavonoids, and phenols
(Table 1). According to Baumgartner et al. (1990) the results of the methanol extract
fractionation of awar-awar leaves contain active compounds in the form of 2 indolizidine
alkaloid that is ficuseptine and antofine, the two compounds that have antifungal and
antibacterial activity. Results of fractionation of ethanol and hexane leaf extract of awar-
awar has potential as an anticancer compound, besides that of leaves, fruits and roots of
awar-awar contain alkaloids, saponins and flavonoids that have the potential as
antimicrobial compounds (Nugroho et al., 2013).
Table 1. Phytochemical test results of combined fraction.
Phytochemicaltest Reaction result Conclusion
Alkaloid chocolatesediment Alkaloid (+)
Triterpenoid yellow to purple Triterpenoid (+)
Phenolat Blackish blue Polyphenol (+)
Flavonoid yellow Flavonoid (+)
Saponin Foamis not Constant Saponin (-)
Tannin No sediment is formed Tannin (-)
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B C D E
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Nduagu et al. (2008) reported the phytochemical content extracts of bark and root bark of
five types of plants can inhibit the growth of Colletotrichum capsici causes anthracnose in
pepper. The extract positive for chemical compounds such as alkaloids (Citrus limon and
Azadirachta indica), tannins (Vernonia amygdalina, Azadirachta indica and Ocimum
gratissimum), glycosides (Vernonia amygdalina, Citrus limon, Azadirachta indica, Ocimum
gratissimum and Anona senegalensis), saponins (Vernonia amygdalina , Citrus limon,
Azadirachta indica, Ocimum gratissimum and Anona senegalensis) and flavonoids
(Azadirachta indica). Lawal et al. (2012) reported methanol and ethyl acetate extracts of the
root bark of Ficus exasperata Vahl. at a concentration of 200 mg / ml can inhibit the growth
of Colletotrichum gloeosporioides with inhibition zone diameter each by 19 mm and 13 mm,
and after phytochemical test, the extract containing saponins and glycosides.
Antifungal Active Compounds Based on GC-MS
Eighteen fractions which showed the highest inhibition to the Colletotrichum acutatum were
combined and then analyzed components contained therein by using GC-MS (GCMS-
QP2010 Ultra SHIMADZU). Chromatogram of the fraction analysis results showed 15 peaks
as shown in Fig. 4, so it is assumed that crude extract of awar-awar leaves may contain a
maximum of 15 active compounds that are antifungal against C. acutatum. Each emerging
peak was further identified by mass spectroscopy, so that each compound has a specific
mass fragmentation pattern.
Figure 4. Chromatogram of GC-MS analysis of the active fractions capable of inhibiting the
growth of C. acutatum.
The identification was done by comparing the mass spectrum of each peak in the mass
spectrum of compounds that are already known to exist in the GC-MS library. Results of the
analysis with GC-MS showed that the active fraction of the leaf extract of awar-awar
contains 14 compounds namely dl-glyceraldehyde dimer, 2,3,5 trimethyl heptane, Sulfurous
acid cyclohexylmethylhexadecyl ester, guanosine, D-Allose, dodecanoic acid methyl ester,
1,2-Benzenedicarboxylic acid diethyl ester, 3-Deoxy-d-mannonic acid, cyclohexane
J. Biol. Chem.
A Research 155 Vol. 34 (1): 150-159 (2017)
B C D E
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tetraethyl 1,2,3,4, (Z) - 9-Tricosene, hexadecanoic acid methyl ester, octadecamethyl-
cyclononasiloxane, 1-Heptacosanol and 1,2-Benzenedicarboxylic acid mono (2-etilhexyl)
ester. Eight of them have been known as antifungal compounds. Those compounds are:
2,3,5 trimethyl heptane, Sulfurous acid cyclohexyl methylhexadecyl ester, dodecanoic acid
methyl ester, 3-Deoxy-d-mannonic acid, hexadecanoic acid methyl ester, octadecamethyl-
cyclononasiloxane, 1 -Heptacosanol and 1,2-Benzenedicar boxylic acid mono (2-etilhexyl)
ester. The specification of each compound contained in the active fraction of the leaf extract
of awar-awar is presented in Table 2.
Table 2. Active compounds that have the potential as a botanical fungicide identified in
the leaf extract of awar-awar based on the analysis with GC-MS.
According to Appuaka et al. (2013) the utility of the benzene compound is the most
importantA thing as a solvent
B and as a rawC material forD making otherE aromatic compounds
which are derivatives of benzene. These compounds act as antioxidants, antifungal and
antimicrobial. Haptane a class of organic compounds alkanes used for petroleum fuels,
solvents, lubricants and industrial raw materials. Akpuaka et al. (2013) reported the heptane
has biological activity as antifungal and antibacterial compounds. Sulfurous acid is an
organic compound containing groups sulfur, are corrosive and hazardous in high
concentrations can cause death. Mazid et al. (2011) reported the Sulfurous acid in higher
plants function as an antifungal compound. Dodecanoic acid is a fatty acid is generally
known as lauric acid. Nakatsuji et al. (2009) reported the lauric acid has biological activity as
antimicrobial compounds. Carolina et al. (2011) reported the dodecanoic acid had biological
activity as an antifungal. 3-Deoxy-D-mannonic acid ester is a compound known as uronat
acid. According to Martinez et al. (2009) uronat acid having biological activity as an
antifungal. Hexadecanoic acid ester compound belonging that is often called sour palmintat.
Hexadecanoic acid compounds have activity as an antioxidant, nemasida, pesticides,
(Murugesan et al., 2013; Elezabeth and Arumugam, 2014). According to Akpuaka et al.
(2013) hexadecanoic acid has activity as antifungal and antibacterial compounds.
Octadecamethyl cyclononasiloxane are compounds that are volatile. Ojekale et al. (2013)
reported the compound octadecamethyl cyclononasiloxane predominantly found in leaf
J. Biol. Chem. Research 156 Vol. 34 (1): 150-159 (2017)
Identification of…………………..……on Chili Pepper Sudirga and Ginantra, 2017
extracts of Thaumatococcus Danielli (Benn.) Benth potential as antioxidants. Dubal et al.
(2013) reported extracts of rhizomes Tectaria coadunata contains octadecamethyl
cyclononasiloxane potentially be used as antioksidaan and antimicrobial. Heptacosanol an
alcoholic, Raman et al. (2012) reported the 1-heptacosanol have biological activity as
nemasida, anticancer, antioxidant and antimicrobial. Benzenedicarboxylic acid ethyl ester is
a compound that is commonly known as monoetilheksil phthalate. Raman et al. (2012)
reported the 1, 2-Benzenedicarboxylic acid had biological activity as antimicrobial,
antioxidant and anticancer. According to Akpuaka et al. (2013) the 1, 2-benzenedicarboxylic
acid compounds have biological activity as antifungal, antibacterial, antiviral and
antioxidant.
CONCLUSION
The leaf extract of awar-awar contains several phytochemicals groups of compounds such
as terpenoids, alkaloids, flavonoids, and phenols. The active fraction of the leaf extract of
awar-awar contains 14 compounds namely dl-glyceraldehyde dimer, 2,3,5 trimethyl
heptane, Sulfurous acid cyclohexylmethylhexadecyl ester, guanosine, D-Allose, dodecanoic
acid methyl ester, 1,2-Benzenedicarboxylic acid diethyl ester, 3-Deoxy-d-mannonic acid,
cyclohexane tetraethyl 1, 2, 3, 4, (Z) - 9- Tricosene, hexadecanoic acid methyl ester,
octadecamethylcyclononasiloxane, 1-Heptacosanol and 1,2-Benzenedicarboxylic acid mono
(2-etilhexyl) ester. Eight of them have been known as antifungal compounds. Those
compounds are: 2,3,5 trimethyl heptane, Sulfurous acid cyclohexyl methylhexadecyl ester,
dodecanoic acid methyl ester, 3-Deoxy-d-mannonic acid, hexadecanoic acid methyl ester,
octadecamethylcyclononasiloxane, 1 -Heptacosanol and 1, 2-Benzenedicar boxylic acid
mono (2-etilhexyl) ester.
ACKNOWLEDGEMENTS
The authors would like to express gratitude to the Rector of Udayana University for the
doctorate program research funding in Postgraduate Program, Faculty of Agriculture
Udayana University and we would like to express to Laboratory of Biopesticide Faculty of
Agriculture Udayana University for providing laboratory’s facilities that made this study
happened.
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Corresponding author: Dr. Sang Ketut Sudirga, Department of Biology, Faculty of
Mathematics and Natural Sciences, Udayana University, Kampus Bukit Jimbaran Bali
Indonesia Email:
[email protected]J. Biol. Chem. Research 159 Vol. 34 (1): 150-159 (2017)
