ISOLATION AND IDENTIFICATION OF SECONDARY METABOLITES OF CHLOROFORM FRACTION OF MACROALGAE Padina australis AS ANTI TUBERCULOSIS
Indonesia Chimica Acta
Vol.7. No. 1, June 2014
Pridawati, et.al.
ISSN 2085-014X
ISOLATION AND IDENTIFICATION OF SECONDARY METABOLITES OF
CHLOROFORM FRACTION OF MACROALGAE Padina australis
AS ANTI TUBERCULOSIS
Eka Pridawati1, Ahyar Ahmad, Usman Hanapi
Department of Chemistry, Natural Sciences Faculty of Hasanuddin University
Abstrak. Penelitian ini bertujuan mengisolasi senyawa metabolit sekunder makroalga Padina
australis fraksi kloroform dan untuk mengetahui konsentrasi minimum dalam menghambat
pertumbuhan Mycobacterium tuberculosis. Metode penelitian yang digunakan melalui
tahapan-tahapan: ekstraksi, isolasi, pemurnian, elusidasi struktur, dan uji bioaktivitas. Dua
senyawa diperoleh dari penelitian ini. Pertama: golongan steroid dan kedua: golongan terpenoid
(senyawa 2) dengan nilai Minimum Inhibition Concentration (MIC) terhadap M. tuberculosis
adalah 400 µ g/mL dan pada konsentrasi 100 µ g/mL, senyawa 2 juga mampu meningkatkan
aktivitas rifampisin 0,5 µg/mL dalam menghambat pertumbuhan bakteri M. Tuberculosis.
Kata Kunci : Anti tuberculosis, identifikasi, isolasi, metabolit sekunder, Padina australis
Abstract. This study aims to isolate the secondary metabolites of macroalgae Padina australis
and the chloroform fraction to determine the minimum concentration to inhibit the growth of
Mycobacterium tuberculosis. The methods used through the stages: extraction, isolation,
purification, structure elucidation, and bioactivity test. In this research, we obtained two
compounds: (i) steroids and (ii) terpenoids (compound 2) with the value of the Minimum
Inhibition Concentration (MIC) against M. tuberculosis was 400 µ g/mL and also at a
concentration of 100 µg/mL. Compound 2 also increased the activity of rifampicin 0,5 µ g/mL in
inhibiting the growth of bacteria M.Tuberculosis.
Keywords: Anti tuberculosis, identification, isolation, Padina australis, secondary metabolites
1
Address for correspondence: [email protected]
1
Indonesia Chimica Acta
Vol.7. No. 1, June 2014
Pridawati, et.al.
INTRODUCTION
Tuberculosis is one of the major
and very serious problem because it was
among the top ten leading cause of death
in the world. Approximately one-third of
the world's population is infected by
M.
tuberculosis.
Every
year,
approximately 8 million people who are
infected develop into active disease and
2
million
died
of
tuberculosis.
Approximately 10% cases of TB infection
in the world is found in Indonesia and puts
it in third place behind India and China.
This was due to the presence of multidrug
resistance M. tuberculosis causes the
condition is getting worse, not only in
Indonesian and even hit around the world,
both developing and developed countries
(WHO, 2009). Therefore, research for new
anti-tuberculosis drugs, effective, and
affordable is necessary to tackle TB
disease, including therapeutic procedures
or administration of anti-TB drugs (new
treatment guidelines).
In the last two decades is estimated
to have been isolated and identified in
2500 secondary metabolites from marine
biota and symbionts, and as many as 353
of them suspected compound has potential
as an ingredient the new of antituberculosis (Mayer, et al., 2011). Extracts
of secondary metabolites from marine
algae contain bioactive compounds that
are known to have pharmacological
activity properties such as antibacterial
(Muniarsih and Rachmaniar, 1999), and
anti-tuberculosis (Copp and Pearce, 2007;
Tasdemir, et a.l, 2007; Azevedo, et al.,
2008; Abdel-Aal,. et al., 2010). So far is
very less even no research data that
explored group of nonpolar compounds
from marine algae and bacterial symbionts
as a raw material of anti-tuberculosis
drugs.
Exploration and marine research
for pharmaceutical use has grown rapidly
within 30-40 years. It is accelerated by
increasing consumer awareness and drug
industry (pharmaceuticals) at home and
ISSN 2085-014X
abroad to prioritize the use of drugs from
natural products known as "back to
nature" (Rismana, 2001).
The results research from Massi
and Ahmad (2011), showed that the red
algae Gelidium amansii of Baranglompo
island waters shown to contain antituberculosis protein compounds that could
inhibit directly or indirectly, the growth of
M. tuberculosis strain H37Rv (ATCC
27394).
Utilization
of
bioactive
compounds will be even better if it can be
obtained from the sources of raw materials
abundant algae and algal species are
relatively abundant in the waters of
Indonesia.
P. australis is one type of brown
algae that have economic value because it
is useful as an animal feed, human food,
fertilizer, and in the health field are useful
as anti-bacterial and anti-microbial.
P. australis, including the type of algae
that is often found in coastal waters of
Indonesia (Atmadja, 1990).
METHODE
Intake and P. australis Hauck Sample
Preparation
Marine algae samples taken in
waters
Selayar
Islands
District.
P. australis sample preparated by wind
dried for isolation purposes.
Extraction and Identification of
Secondary Metabolites
P. australis weighed 3 kg (dry
weight) then macerated with methanol and
then filtered. Extract (filtrate) was
concentrated with a rotary evaporator to
obtain a methanol extract thick.
Furthermore, in bioactivity test against
bacteria Mycobacterium then methanol
extract
P.
australis
liquid-liquid
partitioned with solvents chloroform.
Extracts (filtrate) was concentrated with a
rotary evaporator to obtain chloroform
extract.
2
Indonesia Chimica Acta
Vol.7. No. 1, June 2014
Pridawati, et.al.
ISSN 2085-014X
for 15 minutes. Move again supernatant
into another sterile tube. Next create a
suspension of M. tuberculosis H37Rv in
PBS at No. turbidity standard. 0.5
McFarland. Dilute 1 mL of a suspension
of M. Tuberculosis H37Rv 0.5 McFarland
in 4 mL of PBS in a sterile tube inserted.
Isolation and Purification of Secondary
Metabolites
Partition results using KKV were
fractionated into several fractions in which
the eluent used can be determined based
on the results of the TLC analysis.
Fractions obtained were monitored by
TLC and fractions that have the same Rf
values are merged into one major fraction,
then evaporated to dryness, the specified
weight. Fractionation process is done
repeatedly until pure isolates obtained
using the KKT or KKG with the
appropriate eluent. Isolates was tested by
TLC and tested its bioactivity.
Test
Minimum
Inhibitory
Concentration
(MIC)
against
M. Tuberculosis
4 ml of MGIT medium was added
compounds 2 and 3 with a final
concentration variation in tube 0 µg/mL,
100 µg/mL, 200 µg/mL, dan 400 µg/mL
and rifampicin with end concentration of
the tube 0 μg/mL, 1 μg/mL, and 2 μg/mL.
The tubes are then sealed, shaken, the
outside of the tube is cleaned with
disinfectant tuberkulosidal, and incubated
at 37 °C. Observations were made every
day from 3rd day until 14 th day.
Structure Elucidation of Compounds
The molecular structure of pure
isolate compounds that have antituberculosis maximum bioactivity will be
elucidated by ultraviolet spectroscopy
(UV), Infrared (IR), nuclear magnetic
resonance
(NMR)
and
Gas
Chromatography-Mass
Spectroscophy RESULTS AND DISCUSSION
Compound 1
(GC-MS) (Usman, 2005).
13.41 mg of compound 1 in the
form white powder with TLC shows a
Inoculum M. tuberculosis H37Rv
Culture of M. tuberculosis H37Rv single stain, not fluorescent, visible only
were grown on solid media, aged 14 days, with a reagent stain apparition, the melting
made in Middlebrook 7H9 Broth point of 50-51 °C. The purity of
suspension. Vortex the suspension for compound 1 proved by TLC analysis with
2-3 minutes. The suspension is allowed to three kinds of eluent system that shows a
stand for 20 minutes. Transfer the single stain as shown in Figure 2.
supernatant into a sterile tube and let stand
Figure 1. Chromatogram results of TLC analysis of crystal 1 (compound 1)
3
Indonesia Chimica Acta
Vol.7. No. 1, June 2014
Pridawati, et.al.
ISSN 2085-014X
Spetrokfotometer FTIR spectrum of the compound 1 gave the following as
shown in Figure 2.
Figure 2. IR spectrum of compound 1
Table 1. Absorption peaks IR data of compounds 1 and β-sitosterol (Patra, et al., 2010)
No. Absorption peaks
(cm-1)
3450
1
β-sitosterol (cm-1)
Types of functional groups
3549, 99
-OH stretching vibration alkohol
2
2918 and 2850
2935, 73 and 2867, 38
aliphatic CH stretching vibration
3
1643
1637, 63
C = C stretching vibration
After the test is done by comparison of TLC pure crystals (β-sitosterol) by
Usman (2006) simultaneously, with a single stain showed the same Rf value is 0,63.
Figure 3. KLT Test compounds with β-sitosterol I
4
Indonesia Chimica Acta
Vol.7. No. 1, June 2014
Pridawati, et.al.
Analysis of IR spectra obtained
results in Table 3 do not show a
considerable difference in wavelength
shift. However, the results and data
analysis IR spectroscopy and TLC single
isolates with β-sitosterol is not enough to
conclude that compound 1 is β-sitosterol
considering the melting point of the
compound 1 around 50-51 °C, while the
melting point of β-sitosterol is 139-142 °C
(ChemNet, 2001). Thus compound 1 can
only be inferred based on the results of the
test steroid group (Table 2).
ISSN 2085-014X
Compound 2
43,10 mg of compound 2 as a
white powder with a melting point 49-50
°C and does not fluorescence under UV
light. The results of TLC analysis after
being sprayed with stain and heated
apparition showed elongated single stain
(Figure 4). Class ification test for
compound 2 is shown in Table 4 indicates
that the compound belongs to a class of
steroids.
Figure 4. Chromatogram results of TLC analysis of compound 2
Table 2. Testing class of compounds in each fraction
No.
Extract & Crystals
1
2
3
4
Methanol extracts
CHCl3 extract
Compound 1
Compound 2
Test Group
Terpenoids Steroids Phenolic
+
+
+
+
-
Flavanoid
-
Analysis of FTIR spectrophotometer to compound 2 gave a spectrum as shown
in Figure 5 below.
5
Indonesia Chimica Acta
Vol.7. No. 1, June 2014
Pridawati, et.al.
ISSN 2085-014X
Figure 5. IR spectrum of compound 2
Table 6.The data of IR absorption peaks of compounds 2
No. Absorption peaks (cm-1)
Types of functional groups
1
3452
-OH stretching vibration alkohol
2
2954, 2918 and 2848
aliphatic CH stretching vibration
3
1701
C = O stretching vibration
4
1465
bending - CH
From the results of GC-MS
spectroscopic analysis of the compounds
seen 5 peaks in the chromatogram. Visible
peaks on the chromatogram that have
different characteristics as shown in Table
6, by comparing the GC-MS spectrum of
the data obtained with the standard
compound mass spectrum of the GC-MS
data base (Wiley 275, Lib) is appropriate,
then the expected compound is oxybis(CAS) Dimethyl ether. Based on
spectroscopic data, the IR (KBr) cm-1 υ
maks: at wave number 3452 cm-1 indicates
a stretching vibration (-OH) of an
alcoholic. The existence of absorption
peaks at wave numbers 1701 cm-1 indicate
the presence of carbonyl group (C = O)
carboxylate. Absorption at 2954, 2918,
and 2848 cm-1 shows the presence of
aliphatic CH stretching vibration and the
absorption at 1465 cm-1 indicates the
presence of CH bending (Figure 13), so it
does not match the structure of the data
contained GC-MS with data from FTIR.
So, based on the results of the
classification test and the data from FTIR
can be inferred is a class of terpenoids
with functional groups include OH and
C = O.
Anti-tuberculosis
Activity
of
Compounds 2
MIC test results with variations
rifimpsin concentrations of 0 μg/mL,
1 μg/mL, and 2 μg/mL as well as
variations in the concentration of
compound 2, namely 0 µg/mL, 100
µg/mL, 200 µg/mL, and 400 µg/mL using
6
Indonesia Chimica Acta
Vol.7. No. 1, June 2014
Pridawati, et.al.
MGIT medium can be seen in Table 10.
From these data it can be seen that in the
negative control (P1) of observations to
test
the
MIC
against
bacteria
M. tuberculosis Strains H37Rv. Negative
control no containing compounds 2 and
rifampicin drug (inoculum, OADC, and
the solvent alone) was much overgrown
by bacteria M. tuberculosis H37Rv strains
were characterized by high levels of
flouresensi the MGIT tube. This is
because the bacteria growth on the tube
P1 M. tuberculosis Strain H37Rv no
influence of drugs or compounds that
ISSN 2085-014X
inhibit the growth of two bacteria
M. tuberculosis Strain H37Rv. However,
when the concentration of rifampicin
0,5 µg/mL and compound 2 concentration
of 100 µg/mL (P6) found no bacterial
growth M. tuberculosis Strain H37Rv.
This shows no effect of compound 2 in
inhibiting the growth of bacteria
M. tuberculosis Strain H37Rv. At the
table also shows the growth of
M. tuberculosis H37Rv strain can be
directly inhibited by compounds 2 without
the addition of rifampicin at a concentrat
ion of 400 µg/mL (P10).
Table 9. The effects of addition compound 2 in combination with rifampicin on the
growth of M. tuberculosis H37Rv strain on medium MGIT
Compound
2 (μg/mL)
0
Rifampicin (µg/mL)
0
0,5
1
2
P1
P2
P3
P4
(+ + +)
(+)
(-)
(-)
100
P5
(+ +)
P6
(-)
P7
(-)
P8
(-)
200
P9
(+)
P10
(-)
P 11
(-)
P 12
(-)
400
P1 3
(-)
P14
(-)
P1 5
(-)
P1 6
(-)
inhibiting the
M. Tuberculosis.
growth
Description:
+ + + = TB cell colonies grown much
++
= TB cell colonies grown quite a lot
+
= TB cell colony grows a little
= TB cell colonies did not grow (negative)
CONCLUSION
The
results
showed
that
P. australis contain components secondary
metabolites. Components of secondary
1.
metabolites between another class of
steroids and terpenoids.
Compound 2 (terpenoids) is able to
inhibit
the
growth
of
bacteria
M. Tuberculosis at a concentration of
400 µg/mL and at a concentration of
100 µg/mL was also able to increase the
activity of rifampicin 0,5 µg/mL in
of
bacteria
REFERENCES
1. Abdel-Aal, W.S., Hassan, H.Y.,
Aboul-Fadl, T., and Youssef, A.F.,
2010,
Pharmacophoric
building
models for antituberculosis activity of
the individual Schiff bases of small
combinatorial
library,
Eur.
J.
Med.Chem, 45 (3): 1098-1106.
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Indonesia Chimica Acta
Vol.7. No. 1, June 2014
2.
Pridawati, et.al.
Ahmad, A. and Massi, M.N., 2011,
Anticancer activity of native and
recombinant protein lectins from
marine algae against human HeLa
cancer cells, Inclusive preparation.
3. Atmadja, W.S., 1990, Research and
Development Centre for Oceanology9.
LIPI, Seaweed as medicine Oseana, 17
(1): 1-8, Jakarta.
4. Azevedo, L.G., Muccillo Al-Bai s ch,
Filgueira Dde I, Boyle R, 2008,
Comparative Cytotoxic and AntiTuberculosis Activity of Marine
Sponge Aplysina Caissara Crude
Extracts, Comp Biochem Physiol C
Pharmacol Toxicol, 147 (1): 36-42.
5. ChemNet., 2001, 83-46-5 betasitosterol.
(Online).
(Http://www.chemnet.com/cas/id/8346-5/beta-Sitosterol.html,
Accessed
November 28, 2013)
6. Massi, M.N. and Ahmad, A. , 2011,
Cloning
and
in
vitro
antimycobacterial Activity of Lectin
Proteins
in
Combination
with
Streptomycin to Increase Sensitivity
Against Mycobacterium tuberculosis,
in submittion.
7. Mayer, A.M.S., Rodriguez, A.D.,
Berlinck, R., and Fusetani, N., 2011,
Marine pharmacology in
2007-8:
Marine Compounds with Antibacterial,
Anticoagulants, Antifungal, Antiinflammatory,
antiprotozoal,
antituberculosis
and
Antiviral
Activities; affecting the Immune and
Nervous
System,
and
other
Miscellaneous Mechanisms of Action,
Comparative
Biochemistry
and
Physiology Part C, 153: 191-222.
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8. Muniarsih, T. and Rachmaniar, R.,
1999, Identification of Antimicrobial
Compounds from Sponges Aaptos
Aaptos of Islands Spermonde by Mass
Spectroscopy, Oseon ologi LIPI
Research Center, Jakarta.
Patra, A., Jha, S., Murthy, P.N., Manik,
and Sharone, A., 2010, Isolation and
characterization of stigmast-5-en-3β-ol
(β-sitosterol) from the leaves of
Hygrophila spinosa T. Anders,
International Journal of Pharma
Sciences and Research, 1 (2): 95-100.
10. Rismana, E. , 2001. Potentially Large
Marine Life so Source Raw Materials
Drugs of the Pharmaceutical Industry,
(Online),
(http://Biota20Berpotensi%%%20Laut
20Besar.html, accessed January 25,
2008).
11. Tasdemir, D., Topaloglu, B., Perozzo,
R., Brun, R., O'Neill, R., Carballeira,
N.M., Zhang, X., Tonge, P.J., Linden,
A., and Ruedi, P., 2007, Marine
Natural Products From The Turkish
Sponge Agelas Oroides That Enoyl
Reductases
From
inhibit
the
Plasmodium
falciparum,
Mycobacterium tuberculosis and,
Escherichia coli,15: 6834-6845.
12. Usman, H. , 2005, 2',4'-dihydroxy-3', 5
', 6'-trimetoksicalkon An Anti-Tumor
Compounds of Skin Stem plants
Criptocarya costata (Lauraceae), ITB
Journal of Mathematics and Science,
10: 97-100.
13. World Health Organization, 2009,
Tuberculosis Fact Sheet. Available
from http://www.who.int/mediacentre/
factsheets/fs104/en,
Accessed
on
November
25,
2011.
8
Vol.7. No. 1, June 2014
Pridawati, et.al.
ISSN 2085-014X
ISOLATION AND IDENTIFICATION OF SECONDARY METABOLITES OF
CHLOROFORM FRACTION OF MACROALGAE Padina australis
AS ANTI TUBERCULOSIS
Eka Pridawati1, Ahyar Ahmad, Usman Hanapi
Department of Chemistry, Natural Sciences Faculty of Hasanuddin University
Abstrak. Penelitian ini bertujuan mengisolasi senyawa metabolit sekunder makroalga Padina
australis fraksi kloroform dan untuk mengetahui konsentrasi minimum dalam menghambat
pertumbuhan Mycobacterium tuberculosis. Metode penelitian yang digunakan melalui
tahapan-tahapan: ekstraksi, isolasi, pemurnian, elusidasi struktur, dan uji bioaktivitas. Dua
senyawa diperoleh dari penelitian ini. Pertama: golongan steroid dan kedua: golongan terpenoid
(senyawa 2) dengan nilai Minimum Inhibition Concentration (MIC) terhadap M. tuberculosis
adalah 400 µ g/mL dan pada konsentrasi 100 µ g/mL, senyawa 2 juga mampu meningkatkan
aktivitas rifampisin 0,5 µg/mL dalam menghambat pertumbuhan bakteri M. Tuberculosis.
Kata Kunci : Anti tuberculosis, identifikasi, isolasi, metabolit sekunder, Padina australis
Abstract. This study aims to isolate the secondary metabolites of macroalgae Padina australis
and the chloroform fraction to determine the minimum concentration to inhibit the growth of
Mycobacterium tuberculosis. The methods used through the stages: extraction, isolation,
purification, structure elucidation, and bioactivity test. In this research, we obtained two
compounds: (i) steroids and (ii) terpenoids (compound 2) with the value of the Minimum
Inhibition Concentration (MIC) against M. tuberculosis was 400 µ g/mL and also at a
concentration of 100 µg/mL. Compound 2 also increased the activity of rifampicin 0,5 µ g/mL in
inhibiting the growth of bacteria M.Tuberculosis.
Keywords: Anti tuberculosis, identification, isolation, Padina australis, secondary metabolites
1
Address for correspondence: [email protected]
1
Indonesia Chimica Acta
Vol.7. No. 1, June 2014
Pridawati, et.al.
INTRODUCTION
Tuberculosis is one of the major
and very serious problem because it was
among the top ten leading cause of death
in the world. Approximately one-third of
the world's population is infected by
M.
tuberculosis.
Every
year,
approximately 8 million people who are
infected develop into active disease and
2
million
died
of
tuberculosis.
Approximately 10% cases of TB infection
in the world is found in Indonesia and puts
it in third place behind India and China.
This was due to the presence of multidrug
resistance M. tuberculosis causes the
condition is getting worse, not only in
Indonesian and even hit around the world,
both developing and developed countries
(WHO, 2009). Therefore, research for new
anti-tuberculosis drugs, effective, and
affordable is necessary to tackle TB
disease, including therapeutic procedures
or administration of anti-TB drugs (new
treatment guidelines).
In the last two decades is estimated
to have been isolated and identified in
2500 secondary metabolites from marine
biota and symbionts, and as many as 353
of them suspected compound has potential
as an ingredient the new of antituberculosis (Mayer, et al., 2011). Extracts
of secondary metabolites from marine
algae contain bioactive compounds that
are known to have pharmacological
activity properties such as antibacterial
(Muniarsih and Rachmaniar, 1999), and
anti-tuberculosis (Copp and Pearce, 2007;
Tasdemir, et a.l, 2007; Azevedo, et al.,
2008; Abdel-Aal,. et al., 2010). So far is
very less even no research data that
explored group of nonpolar compounds
from marine algae and bacterial symbionts
as a raw material of anti-tuberculosis
drugs.
Exploration and marine research
for pharmaceutical use has grown rapidly
within 30-40 years. It is accelerated by
increasing consumer awareness and drug
industry (pharmaceuticals) at home and
ISSN 2085-014X
abroad to prioritize the use of drugs from
natural products known as "back to
nature" (Rismana, 2001).
The results research from Massi
and Ahmad (2011), showed that the red
algae Gelidium amansii of Baranglompo
island waters shown to contain antituberculosis protein compounds that could
inhibit directly or indirectly, the growth of
M. tuberculosis strain H37Rv (ATCC
27394).
Utilization
of
bioactive
compounds will be even better if it can be
obtained from the sources of raw materials
abundant algae and algal species are
relatively abundant in the waters of
Indonesia.
P. australis is one type of brown
algae that have economic value because it
is useful as an animal feed, human food,
fertilizer, and in the health field are useful
as anti-bacterial and anti-microbial.
P. australis, including the type of algae
that is often found in coastal waters of
Indonesia (Atmadja, 1990).
METHODE
Intake and P. australis Hauck Sample
Preparation
Marine algae samples taken in
waters
Selayar
Islands
District.
P. australis sample preparated by wind
dried for isolation purposes.
Extraction and Identification of
Secondary Metabolites
P. australis weighed 3 kg (dry
weight) then macerated with methanol and
then filtered. Extract (filtrate) was
concentrated with a rotary evaporator to
obtain a methanol extract thick.
Furthermore, in bioactivity test against
bacteria Mycobacterium then methanol
extract
P.
australis
liquid-liquid
partitioned with solvents chloroform.
Extracts (filtrate) was concentrated with a
rotary evaporator to obtain chloroform
extract.
2
Indonesia Chimica Acta
Vol.7. No. 1, June 2014
Pridawati, et.al.
ISSN 2085-014X
for 15 minutes. Move again supernatant
into another sterile tube. Next create a
suspension of M. tuberculosis H37Rv in
PBS at No. turbidity standard. 0.5
McFarland. Dilute 1 mL of a suspension
of M. Tuberculosis H37Rv 0.5 McFarland
in 4 mL of PBS in a sterile tube inserted.
Isolation and Purification of Secondary
Metabolites
Partition results using KKV were
fractionated into several fractions in which
the eluent used can be determined based
on the results of the TLC analysis.
Fractions obtained were monitored by
TLC and fractions that have the same Rf
values are merged into one major fraction,
then evaporated to dryness, the specified
weight. Fractionation process is done
repeatedly until pure isolates obtained
using the KKT or KKG with the
appropriate eluent. Isolates was tested by
TLC and tested its bioactivity.
Test
Minimum
Inhibitory
Concentration
(MIC)
against
M. Tuberculosis
4 ml of MGIT medium was added
compounds 2 and 3 with a final
concentration variation in tube 0 µg/mL,
100 µg/mL, 200 µg/mL, dan 400 µg/mL
and rifampicin with end concentration of
the tube 0 μg/mL, 1 μg/mL, and 2 μg/mL.
The tubes are then sealed, shaken, the
outside of the tube is cleaned with
disinfectant tuberkulosidal, and incubated
at 37 °C. Observations were made every
day from 3rd day until 14 th day.
Structure Elucidation of Compounds
The molecular structure of pure
isolate compounds that have antituberculosis maximum bioactivity will be
elucidated by ultraviolet spectroscopy
(UV), Infrared (IR), nuclear magnetic
resonance
(NMR)
and
Gas
Chromatography-Mass
Spectroscophy RESULTS AND DISCUSSION
Compound 1
(GC-MS) (Usman, 2005).
13.41 mg of compound 1 in the
form white powder with TLC shows a
Inoculum M. tuberculosis H37Rv
Culture of M. tuberculosis H37Rv single stain, not fluorescent, visible only
were grown on solid media, aged 14 days, with a reagent stain apparition, the melting
made in Middlebrook 7H9 Broth point of 50-51 °C. The purity of
suspension. Vortex the suspension for compound 1 proved by TLC analysis with
2-3 minutes. The suspension is allowed to three kinds of eluent system that shows a
stand for 20 minutes. Transfer the single stain as shown in Figure 2.
supernatant into a sterile tube and let stand
Figure 1. Chromatogram results of TLC analysis of crystal 1 (compound 1)
3
Indonesia Chimica Acta
Vol.7. No. 1, June 2014
Pridawati, et.al.
ISSN 2085-014X
Spetrokfotometer FTIR spectrum of the compound 1 gave the following as
shown in Figure 2.
Figure 2. IR spectrum of compound 1
Table 1. Absorption peaks IR data of compounds 1 and β-sitosterol (Patra, et al., 2010)
No. Absorption peaks
(cm-1)
3450
1
β-sitosterol (cm-1)
Types of functional groups
3549, 99
-OH stretching vibration alkohol
2
2918 and 2850
2935, 73 and 2867, 38
aliphatic CH stretching vibration
3
1643
1637, 63
C = C stretching vibration
After the test is done by comparison of TLC pure crystals (β-sitosterol) by
Usman (2006) simultaneously, with a single stain showed the same Rf value is 0,63.
Figure 3. KLT Test compounds with β-sitosterol I
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Pridawati, et.al.
Analysis of IR spectra obtained
results in Table 3 do not show a
considerable difference in wavelength
shift. However, the results and data
analysis IR spectroscopy and TLC single
isolates with β-sitosterol is not enough to
conclude that compound 1 is β-sitosterol
considering the melting point of the
compound 1 around 50-51 °C, while the
melting point of β-sitosterol is 139-142 °C
(ChemNet, 2001). Thus compound 1 can
only be inferred based on the results of the
test steroid group (Table 2).
ISSN 2085-014X
Compound 2
43,10 mg of compound 2 as a
white powder with a melting point 49-50
°C and does not fluorescence under UV
light. The results of TLC analysis after
being sprayed with stain and heated
apparition showed elongated single stain
(Figure 4). Class ification test for
compound 2 is shown in Table 4 indicates
that the compound belongs to a class of
steroids.
Figure 4. Chromatogram results of TLC analysis of compound 2
Table 2. Testing class of compounds in each fraction
No.
Extract & Crystals
1
2
3
4
Methanol extracts
CHCl3 extract
Compound 1
Compound 2
Test Group
Terpenoids Steroids Phenolic
+
+
+
+
-
Flavanoid
-
Analysis of FTIR spectrophotometer to compound 2 gave a spectrum as shown
in Figure 5 below.
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Indonesia Chimica Acta
Vol.7. No. 1, June 2014
Pridawati, et.al.
ISSN 2085-014X
Figure 5. IR spectrum of compound 2
Table 6.The data of IR absorption peaks of compounds 2
No. Absorption peaks (cm-1)
Types of functional groups
1
3452
-OH stretching vibration alkohol
2
2954, 2918 and 2848
aliphatic CH stretching vibration
3
1701
C = O stretching vibration
4
1465
bending - CH
From the results of GC-MS
spectroscopic analysis of the compounds
seen 5 peaks in the chromatogram. Visible
peaks on the chromatogram that have
different characteristics as shown in Table
6, by comparing the GC-MS spectrum of
the data obtained with the standard
compound mass spectrum of the GC-MS
data base (Wiley 275, Lib) is appropriate,
then the expected compound is oxybis(CAS) Dimethyl ether. Based on
spectroscopic data, the IR (KBr) cm-1 υ
maks: at wave number 3452 cm-1 indicates
a stretching vibration (-OH) of an
alcoholic. The existence of absorption
peaks at wave numbers 1701 cm-1 indicate
the presence of carbonyl group (C = O)
carboxylate. Absorption at 2954, 2918,
and 2848 cm-1 shows the presence of
aliphatic CH stretching vibration and the
absorption at 1465 cm-1 indicates the
presence of CH bending (Figure 13), so it
does not match the structure of the data
contained GC-MS with data from FTIR.
So, based on the results of the
classification test and the data from FTIR
can be inferred is a class of terpenoids
with functional groups include OH and
C = O.
Anti-tuberculosis
Activity
of
Compounds 2
MIC test results with variations
rifimpsin concentrations of 0 μg/mL,
1 μg/mL, and 2 μg/mL as well as
variations in the concentration of
compound 2, namely 0 µg/mL, 100
µg/mL, 200 µg/mL, and 400 µg/mL using
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Pridawati, et.al.
MGIT medium can be seen in Table 10.
From these data it can be seen that in the
negative control (P1) of observations to
test
the
MIC
against
bacteria
M. tuberculosis Strains H37Rv. Negative
control no containing compounds 2 and
rifampicin drug (inoculum, OADC, and
the solvent alone) was much overgrown
by bacteria M. tuberculosis H37Rv strains
were characterized by high levels of
flouresensi the MGIT tube. This is
because the bacteria growth on the tube
P1 M. tuberculosis Strain H37Rv no
influence of drugs or compounds that
ISSN 2085-014X
inhibit the growth of two bacteria
M. tuberculosis Strain H37Rv. However,
when the concentration of rifampicin
0,5 µg/mL and compound 2 concentration
of 100 µg/mL (P6) found no bacterial
growth M. tuberculosis Strain H37Rv.
This shows no effect of compound 2 in
inhibiting the growth of bacteria
M. tuberculosis Strain H37Rv. At the
table also shows the growth of
M. tuberculosis H37Rv strain can be
directly inhibited by compounds 2 without
the addition of rifampicin at a concentrat
ion of 400 µg/mL (P10).
Table 9. The effects of addition compound 2 in combination with rifampicin on the
growth of M. tuberculosis H37Rv strain on medium MGIT
Compound
2 (μg/mL)
0
Rifampicin (µg/mL)
0
0,5
1
2
P1
P2
P3
P4
(+ + +)
(+)
(-)
(-)
100
P5
(+ +)
P6
(-)
P7
(-)
P8
(-)
200
P9
(+)
P10
(-)
P 11
(-)
P 12
(-)
400
P1 3
(-)
P14
(-)
P1 5
(-)
P1 6
(-)
inhibiting the
M. Tuberculosis.
growth
Description:
+ + + = TB cell colonies grown much
++
= TB cell colonies grown quite a lot
+
= TB cell colony grows a little
= TB cell colonies did not grow (negative)
CONCLUSION
The
results
showed
that
P. australis contain components secondary
metabolites. Components of secondary
1.
metabolites between another class of
steroids and terpenoids.
Compound 2 (terpenoids) is able to
inhibit
the
growth
of
bacteria
M. Tuberculosis at a concentration of
400 µg/mL and at a concentration of
100 µg/mL was also able to increase the
activity of rifampicin 0,5 µg/mL in
of
bacteria
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