Secondary Metabolites on Morus macroura Miq 509 Hairy Root Cultures with the Addition of the
Saccharomyces cerevisiae Yeast Extract Elicitor
Nizar Happyana, Euis Holisotan Hakim
Natural Product Chemistry Group, Study Program of Chemistry, Institut Teknologi Bandung
Abstract
Morus macroura Miq. locally known as Andalas, is a rare Indonesian endemic plant which is reported to contain several interesting secondary
metabolites such as chalcomoracin which possesses antimicrobial activity, guangsanon I and J having the anti-inflammatory and antioxidant activites
and andalasin A (4) having antinematodal and antifungal activities. Plant tissue culture is one of the techniques that can handle the problem in further
investigation on this endangered potencial plant. The transformation of the plant nucleus by Agrobacterium rhyzogenes resulted the hairy root which
can produce secondary metabolites in the short time. The aim of this investigation is to isolate and determine the structure of secondary metabolites
and study the effect of the addition of the S. cerevisiae yeast extract elicitor toward the production of secondary metabolites on M. macroura Miq 509
hairy roots cultures. From 35 g of the hairy root of M. macroura Miq 509 with the addition of the Saccharomyces cerevisiae yeast extracts elicitor,
yielded four Diel Alder type adduct compounds, namely M1 (mulberrofuran P; 29 mg), M2 (chalcomoracin; 193 mg), M4 (20 mg), M7 (5 mg), an
aliphatic compound, namely M3 (Intan 8; 3 mg); and two unknown aromatic compounds [M5 (9 mg); M6 (5 mgThe structures of M1-M3 were
determined based on UV, IR spectra and compared with the standard data and M4 and M7 were determined based on spectroscopic data including
NMR, MS, UV, and IR. M2, M4 and M7 were showed potent anticancer activity against human ovarian cancer cell lines (OVCAR-3) (IC50: 11; 11;
and 12 μg/ml). Based on HPLC analysis and bioactivity assay toward sample of the methanol extract and control of the methanol extract, the addition
of the S. cerevisiae yeast extract elicitor on M. macroura Miq 509 hairy root cultures caused increase of production of secondary metabolites which
possess activity against OVCAR-3.
Introductions
Morus macroura Miq locally known as ‘Andalas’, is a rare Indonesian
plant that has good potency as source of drugs. This plant is found
mainly in the province of West Sumatra, Indonesia. It is a tree
growing up to 35 m and found at altitudes of 900-1600 m (Hayne,
1987). In the region, the plant has been valued for the high quality of
the wood due to its resistance to insects (Hayne, 1987). M. macroura
Miq has secondary metabolites which exhibit interesting biological
properties, such as chalcomoracin (12) which possesses antimicrobial
activity (Fukai 2005), guangsangon O (6) having the antioxidant
activity (Dai, 2006), guangsanon I (7) and J (8) having the antiinflammatory and antioxidant activites (Dai, 2004), andalasin A (4)
having antinematodal and antifungal activities (Syah, 2000).
Unfortunately, the destruction of Indonesian forest threatens the
existence and future study of this rare plant, especially the study of
secondary metabolites.
Tissue culture is a method to solve the problems of future secondary
metabolites study of M. macroura Miq. This technique could quickly
produce M. macroura Miq plants as well as secondary metabolites.
The recent researches which have been done by Research Group of
Natural Product Chemistry, ITB, were isolated lupeol acetate,
isobavachalcon, kuwanon J, chalcomoracin (Maisyarah, 2004),
morachalcon A (Agustina, 2003), mulberrofuran P (Nelson, 2004),
kuwanon R and kuwanol E (Marlina, 2003; Sartika, 2004) from tissue
cultures of M. macroura Miq. Hairy roots cultures of M. macroura
Miq is one of the techniques of tissue cultures to produce the roots and
the secondary metabolites in a short time. In previous researches,
some compounds have been isolated from M. macroura Miq hairy
roots by Research Group of Natural Product Chemistry, ITB. The next
step of this research is to increase the secondary metabolites
production. It is a breakthrough step, because it can produce the
secondary metabolites in a large scale. Addition of an elicitor in tissue
cultures is one of the techniques to pursue this step. In this research,
the Saccharomyces cereviciae yeast extracts elicitor was added on
hairy root cultures of M. macroura Miq. It is expected to increase the
secondary metabolites production. Objectives of this research was to
isolate and determine the structure of secondary metabolites from M.
macroura Miq 509 hairy roots which was added S. cerevisiae yeast
extract elicitor and study the effect of addition S. cerevisiae yeast
extract elicitor toward the production of secondary metabolites on M.
macroura Miq 509 hairy roots cultures.
Experiments
Cultures of S. cerevisiae and Preparation the Elicitor
S. cerevisiae yeast was obtained from Microbiology Laboratory,
High School of Life Science, ITB. The germ yeast was
propagated on a solid glucose yeast extract (GYE) media in room
temperature. After two days, it was transferred into a liquid GYE
media and shaken in a shaker machine (100 rpm). Every two
days, the S. cerevisiae liquid culture was proliferated until a
sufficient quantity for harvested. The yeast cultures were
harvested at the beginning of the age of stationery phase (16
hours). After that, it was autoclaved at temperature 121 ˚C and
pressure 15 psi during 15 minutes, centrifuged at 5000 rpm on a
shaker machine during 10 minutes. The supernatants part was
removed and the cells were washed twice using sterile water,
dried in an oven at temperature 50 °C until a constant weight, and
grinded to powders. In this research, the concentration of S.
cerevisiae yeast extract elicitor is 2.5 % w/v. It was made with
dissolve a 2.5 gram of S. cerevisiae yeast extract in 100 ml of the
sterile water, and then it was autoclaved at temperature 121 ˚C
and pressure 15 psi during 15 minutes.
Hairy Root Cultures of M. macroura Miq 509
Media for growing hairy root cultures was a Murashige Skoog
(MS) media which modified with addition of 0.5 ppm of indol-3buteric acid (IBA). M. macroura Miq. 509 hairy roots sample was
obtained on an inactive form from the previous researchers. It
was activated in a liquid media. The subculture was carried out
every 3-4 weeks until a sufficient quantity. During 6 months, 85
flasks of the M. macroura Miq. 509 hairy root cultures were
yielded. Two of them were controls, and the others were samples.
When the 83 flasks of M. macroura Miq. 509 hairy root cultures
at stationery phase, (8 weeks), 0.5 ml of the S. cerevisiae yeast
extract elicitor (2.5% w/v) was added on every flask, and
incubated during 7 days. After that, the hairy roots were
separated from liquid media, dried in the room temperature, and
grinded to powders. The yield was 35 gram of powders sample of
hairy roots of M. macroura Miq. 509. The control of M.
macroura Miq. 509 hairy root cultures at the age of 8 weeks were
separated from liquid media, dried in the room temperature, and
grinded to powders.
Isolations and Characterizations of Secondary Metabolites
All pure compounds were checked with UV and FTIR spectrometers.
UV spectra were performed on a Varian Corry 100 Conc spectrometer
with methanol as solvents, NaOH and AlCl3 as shifting reagents. FTIR
spectra were carried out on a FTIR spectrum ONE Perkin Elmer
spectrometer with KBr as pellets. The FTIR spectra were compared
with FTIR standard database in Natural Product Chemistry
Laboratory, ITB. Be sides checked with UV and FTIR spectrometers,
M4 and M7 were checked with MS and NMR spectrometers. NMR
spectra were recorded on JEOL JNM A 5000 at 400 MHz ( 1H) and
100 MHz (13C). MS spectra were obtained on MS spectrometer.
Isolation of secondary metabolites comprises maceratations,
fractionations and purifications using some chromatography methods,
namely liquid vacuum chromatography, radial chromatography, flash
chromatography, sephadex column and a column chromatography.
The information of isolations can be looked at figure 1.
HPLC Analysis
HPLC analysis was performed using a Shimadzu-VP system
(Shimadzu, s’-Hortengenbosch, The Netherlands) consisting of a LC10AT pomp, a Kontron 360 auto sampler, a SPD-M10A DAD
detector, a FCV-10AL low pressure gradient mixer, a SCL-10A
system controller, a FIAtron systems CH-30 column heater, operated
with CLASS-VP software, version 6.12SP4. The column used was a
Licrosphere 5 RP-18 (250 x 4 mm; Chrompack, Middelburg, the
Netherlands). The injection volume was 20 μl with a flow rate of 1 ml
mim-1 using a time program of 45 minutes. The mobile phase was
consisted of Water (0.05% formic acid) (solvent A) and MeCN
(0.05% formic acid) (solvent B) and used a linear gradient system with
given time endpoints: 0.01 min: 5% B; 15 min: 30% B; 30 min: 95%
B; 35 min: 95% B; 40 min: 5% B; and 45 min: 5% B. For HPLC
analysis, the sample of extract methanol was prepared with
dissolving 1 mg sample in 1 ml methanol, transferred into a micro
tube and closed immediately, and subjected into a HPLC
instrument. This procedure was also applied toward the control of
extract methanol.
Bioactivity Assay of OVCAR-3 Cells
In this research, bioactivity assay was carried out with human
ovarian cancer cell line (OVCAR-3) assay. It was applied toward
M2, M4, M7, the sample of extract methanol of hairy root
cultures of M. macroura Miq 509 which added the elicitor of
S.cerevisiae yeast extract, and the control of extract methanol of
hairy root cultures of M. macroura Miq 509. The OVCAR-3 cells
were growth on Dulbecco's Modified Eagle Medium (D-MEM)
which is contains 4500 mg/L glucose, 4mM L-glutamine and 110
mg/L sodium pyruvate. This assay was carried out on plates
containing 96 wells. Taxol was used as a control. 100 μl cells
(5000 cells) plated into each well, incubated through 1 day. 1 mg
sample was diluted into 1 ml methanol, evaporated until dried,
redissolved with 10 μl DMSO and added 990 μl DMEM medium.
The samples were made with various concentrations by dilutions.
Every sample (100 μl) was added into wells containing OVCAR3 cells (100 μl) and incubated during 4 days. After the incubation
phase, the bioactivity of each sample was checked with CellTiter
96® AQueous One Solution Cell Proliferation Assay. 50 μl of the
solution was added into each well using a multi channel pipette,
incubated during for 4 hours at 37°C in a humidified, 5% CO 2
atmosphere. After that, the absorbance was recorded at 490 nm
using a 96-well plate reader, and then the bioactivity was
calculated.
Dried powdereed of
M. macroura Miq 509 hairy roots (35 gram)
Maceration with MeOH
MeOH extract (7.9 gram)
Fractionation with liquid vacuum chromatography
Fraction A
87 mg
M3
3 mg
M2
3 mg
Fraction B
258 mg
Fraction C
418 mg
Fraction D
636 mg
M2
161 mg
M1
21 mg
M4
20 mg
M4
20 mg
M4
20 mg
M4
20 mg
Fraction E
58 mg
M7
5 mg
Fraction F
67 mg
FractionG
191 mg
M1
6 mg
Fraction H
67 mg
MMSC2
4 mg
Figure 1 Isolation scheme of secondary metabolites on M. macroura Miq of hairy roots
Results and Discussions
According to table 1, M1 possessed 95% similarity with
mulberrofuran P, M2 possessed 95% similarity with chalcomoracin,
and M3 possessed similarity 93% with Intan 8. Therefore It suggested
that M1 is mulberofuran P, M2 is chalcomoracin, and M3 is Intan 8,
namely an aliphatic compound.
The HRFAB mass spectrum of M4 showed a quasi molecular ion peak
at m/z 649.5 [M + H]+, consistent with a molecular formula C39H36O9.
The 1H-NMR spectrum of M4 exhibited proton signals as follows; a
singlet at δ 12.95 (1H, s, chelated OH), assignable to a hydrogen
bonded hydroxyl group; the proton signals at δ 6.92(1H, brs, H3), δ 6.92 (1H, brs, H-5), and δ 7.34 (1H, d, 8.1), were assigned as
a 2-phenylbenzofuran (rings A and B); one set of ABX aromatic
protons at δ 6.30 (1H, dd, 8.4, 2.2, H19”), δ 6.50 (1H, d, 2.2, H17”), and δ 6.98 (1H, d, 8.4, H-20”) assignable to a 2,4disubstituted phenyl ring (ring F); the proton signals at δ 6.76
(1H, brs, H-2’), and δ 6.76 (1H, brs, H-6’) were assigned as a
symmetrical 3,4,5-trisubstituted phenyl ring (ring C); one set of
AB aromatic protons at δ 6.43 (1H, d, 9.32, H-13”), and δ 8.44
(1H, d, 9.3, H-14”) assignable to ring E; one set of γ,γ-dimethylallyl
group was assigned by proton signals at δ δ 1.59 (3H, s, H-25”), δ 1.70
(3H, s, H-24”), 3.25 (1H, d, 7.0, H-21”), and δ 5.14 (1H, t, 7.0, H22”); and protons attributable to a trisubtituted methylcyclohexene
ring at δ 1.95 (3H, s, H-7”), δ 2.48 (1H, m, H-6”), δ 2.17 (1H, m, H6”), δ 3.74 (1H, t, 2.92, H-5”), δ 4.10 (1H, br s, H-3”), δ 4.63 (t, 4.8,
H-4”), and δ 5.76 (1H, br s, H-2”).
The 1H- and 13C-NMR spectral data of M4 displayed similar
substituted moieties to those chalcomoracin except for the difference
in methylcyclohexene ring chemical shift. Chalcomoracin is a
compound of Diels Alder type adduct which possesses relative
configurations of a cis-trans adduct, and possesses the absolute
configurations as S (C-3”), R (C-4”), and S (C-5”) (Takasugi, 1980).
Chalcomoracin was possessed an epimer, namely mongolicin F which
was isolated from M. mongolica (Kang, 2005). Mongolicin F is a
compound of Diels Alder type adduct which possesses relative
configurations of an all trans adduct, and possesses the absolute
configurations as R (C-3”), S(C-4”), and R (C-5”) (Kang, 2005). The
methylcyclohexene ring chemical shift of M4 is different with
methylcyclohexene ring chemical shift of mongolicin F and
chalcomoracin. It was suggested that their different caused by the
different in configurations. Therefore, M4 was suggested as a
compound of Diels Alder type adduct which similar with
chalcomoracin, possesses relative configurations of a cis-cis adduct,
and possesses the absolute configurations as S (C-3”), R (C-4”), and R
(C-5”).
λ max of
UV spectra
(nm)
203, 277,
323
Compounds
Weight
(mg)
M1
29
M2
193
M3
3
M4
20
203, 319
M5
9
203, 225,
282, 323
M6
5
205, 319,
398
M7
5
205, 276,
332
203, 319,
333
-
Absorptions
band of IR
spectra (cm-1)
3375, 2972,
2920, 1619,
1489, 1432
3349, 2913,
1669, 1619,
1489, 1380
3329, 2919,
2850, 1734,
1622, 1464,
1380
3436, 2913,
1622, 1464,
1380
3445, 2927,
1619, 1497,
1461
3440, 2920,
1621, 1490,
1443
3439, 2920,
1614, 1508,
1452
Yield of IR spectra
comparison
Mulberrofuran P
(95%)
Chalcomoracin
(95%)
Intan 8 (93%)
-
-
-
-
Table 1. Data of IR and UV spectra of M1-M7
No.
2
3
3a
4
5
6
7
7a
1’
2’
3’
4’
5’
6’
δH, (multiplicity, J in Hz)
6.92 (br s)
7.34 (d, 8.4)
6.76 m
6.92 (br s)
6.76 (br s)
6.76 (br s)
δC
156.4
101.8
122.5
121.8
113.1
157.9
98.3
155.3
130.9
103.4
156.5
115.8
156.5
103.4
1’’
2’’
3’’
4’’
5’’
6’’
7’’
8’’
9’’
10’’
11’’
12’’
13’’
14’’
15’’
16’’
17’’
18’’
19’’
20’’
21’’
22’’
5.76 (br s)
4.10 (br s)
4.63 (t, 4.8)
3.74 (t, 2.92)
2.48 m, 2.17 m
1.95 (3H, s)
6.43 (d, 9.32)
8.44(d, 9.3)
6.50 (d, 2.2)
6.30 (dd, 8.4, 2.2)
6.98 (d, 8.4)
3.25 (2H, d, 7.0)
5.14 (t, 7.0)
133.7
124.3
33.1
47.7
25.3
32.1
23.8
209.8
113.3
164.6
116.6
163.2
108.1
132.1
121.8
156.6
103.4
157.8
107.4
128.7
22.1
123.0
Table 2. 1H (400 MHz) and 13C-NMR (100 MHz) data of M 4 in d6-acetone
M7 has a quasi-molecular formula of C39H36O8 deduced by
HRFABMS (m/z = 633.5 [M + H]+). The NMR properties of M7,
including COSY and HMQC spectra and spin decoupling
experiments, suggested that M7 is a ketalized Diels-Alder type
adduct. The 1H-NMR spectrum of M7 exhibited proton signals of
a set of AB aromatic protons at δ 6.34 (1H, d, 8.4, H-5), and δ
7.38 (1H, d, 8.4, H-6) which were assigned as a 2,3,4-trisubtituted
phenyl ring (ring A). It was confirmed by HMBC spectra which
were showed correlations between H-5, C-1, and C-4; and H-6,
C-2, and C-4 on ring A. The proton signals at δ 6.63 (1H, br s, H2’), and δ 6.65 (1H, br s, H-6’) were assigned as a 3,4,5trisubtituted phenyl ring (ring B), and it was confirmed by HMBC
spectra. Two set of ABX aromatic protons at δ 6.36 (1H, d, 2.6,
H-17’’), δ 6.51 (1H, dd, 8.4, 2.6, H-19’’), and δ 7.13 (1H, d, 8.4,
H-20’’) were assigned as a 2,4-disubtituted phenyl ring (ring E)
and δ 6.41 (1H d 8.4, H-11”), δ 6.35 (1H, dd, 8.8, 2.6, H-13”),
and δ 7.15 (1H, d, 8.8, H-14”) were assigned as a 2,4-disubtituted
phenyl ring (ring G). These ABX aromatics were confirmed by
HMBC spectra. The proton signals at δ 6.86 (1H, d, 16.5, H-8)
and δ 7.30 (1H, d, 16.5, H-7) were assigned as a trans 1,2substituted ethenyl of a stilbene, and it was confirmed by HMBC
spectra which were showed correlations between H-7, C-2, C-6,
and C1’; and H-8, C-1, C-6, C-2’, and C-6’. The γ,γ-dimethylallyl
group was assigned by proton signals at δ 1.58 (3H, s, H-25), δ
1.71 (3H, s, H-24), δ 3.32 (1H, d, 7.0, H-21), and δ 5.20 (1H, t,
7.0, H-22), and it was confirmed by HMBC spectra. The proton
signals at δ 1.77 (3H, s, H-7”), δ 2.70 (1H, dd, 17.0, 4.8, H-6”), δ
2.00 (1H, overlapped, H-6”), δ 2.96-2.98 (1H, m, H-4”), δ 2.962.98 (1H, m, H-5”), δ 3.63 (1H, br t, 5.4, H-3”), and δ 6.44 (1H,
br d, 5.4, H-2”) were assigned as a trisubtituted
methylcyclohexene ring and it was confirmed by COSY and
HMBC spectra. The M7 proton signals of trisubtituted
methylcyclohexene ring was similar with sorecin I which has the
absolute configurations of the protons as S (C-3”), R (C-4”), and S
(C-5”)
(Ferrari,
2001).
Therefore,
the
trisubtituted
methylcyclohexene ring of M7 was suggested as S (C-3”), R (C4”), S (C-5”), and the relative configurations of chiral centers on
methylcyclohexane ring of M7 was suggested as a cis-trans
adduct (3”-4”-cis, 4”-5”-trans). According to IR, UV, MS, and
NMR spectra, M7 was suggested as a ketalized Diels-Alder type
adduct which is source from a chalcone and a
dehydroprenylstilbene.
6.65 (1H, br s)
δC
116.9
157.5
117.0
159.1
107.1
127.9
124.6
125.3
139.4
106.9
157.6
111.6
156.9
107.5
1’’
2’’
3’’
4’’
6.44 (1H, br d, 5.4)
3.63 (1H, br t, 5.4)
2.96-2.98 (1H, m)
133.5
122.8
35.4
37.2
5’’
2.96-2.98 (1H, m)
28.6
6’’
2.70 (1H, dd, 17.0, 4.8),
2.00 (1H, overlapped)
1.77 (3H, s)
35.9
No
1
2
3
4
5
6
7
8
1’
2’
3’
4’
5’
6’
7’’
8’’
9’’
10’’
11’’
12’’
13’’
14’’
15’’
16’’
17’’
18’’
19’’
20’’
21’’
δH, (multiplicity, J in Hz)
6.34 (1H, d, 8.4)
7.38 (1H, d, 8.4)
7.30 (1H, d, 16.5)
6.86 (1H, d, 16.5)
6.63 (1H, br s)
6.41 (1H d 8.4)
6.35 (1H, dd, 8.8, 2.6)
7.15 (1H, d, 8.8)
6.36 (1H, d, 2.6)
6.51 (1H, dd, 8.4, 2.6)
7.13 (1H, d, 8.4)
3.32 (1H, d, 7.0)
23.8
103.6
117.0
157.6
103.5
157.5
108.3
126.1
117.2
152.6
103.8
157.5
110.2
128.3
22.9
22’’
23’’
24’’
5.20 (1H, t, 7.0)
1.71 (3H, s)
123.8
131.2
17.9
25’’
1.58 (3H, s)
25.8
HMBC, COSY
Peaks Areas
C4
C2, C4, C7
C2, C6, C1’
C1, C6
C8, C3’, C4’, C6’
C8, C1’, C2’, C4’,
C5’
C6’’
C2’’
C2’’, C3’’, C5’’,
C6’’
C2’’, C3’’, C4’’,
C6’’
C4’’, C5’’
C1’’, C2’’, C6’’
C10’’, C12’’,
C13’’
C9’’
C8’’, C10’’, C12’’
C15’’, C16’’
C18’’
C16’, C18’
C3, C4, C22’’,
C23’’
C21’’, C23’
C22’’, C23’’,
C25’’
C22’’, C23’’,
C24’’
Table 3. 1H (400 MHz) and 13C-NMR (100 MHz) data of M 7 in d6-acetone
The Effect of the Addition of the S.cerevisiae yeast extract
elicitor on M.macroura Miq 509 hairy root cultures
Some compounds on the sample of the methanol extract were
produced with concentration higher than on the control of the
methanol extract (see table 4). The concentrations of compounds E, H
and I on the sample of extract methanol, significantly higher (more or
less: 13 times, 10 times, and 22 times) than on the control of methanol
extract. Otherwise, M1, M2, M5, M6, and M7 on the sample were
produced with concentrations higher (more or less: 7 times, 1.6 times,
4 times, 3 times, and 3 times) than on the control. Other compounds
on the sample which were produced with concentrations higher than
on the control were compounds A, C, J, and Q (more or less: 3 times,
1.5 times, 3 times, and 2 times). Other side, there was a compound
which was discovered on the sample of the methanol extract and it
was not found on the control of methanol extract, namely compound
D. All of these was indicated that the addition of the elicitor was
quantitatively increased some secondary metabolites productions.
Compou
nds
Retention
time
(minutes)
A
B
C
D
E
F (M5)
G (M7)
H
I
J
K (M1)
L (M3)
N (M2)
O
P (M6)
Q
R
S
T
U
10.987
14.315
25.792
26.411
26.741
26.976
27.672
28.224
28.469
28.789
29.045
29.237
30.197
31.168
32.075
33.897
20.800
23.589
24.233
29.696
Sample of
the methanol
extract
Control of
the methanol
extract
Sample of
the media
extract
3484
1475
8793
5367
12968
7507
7069
49053
29641
24244
15594
172364
9546
136973
2576
360453
22213
32853
9152
2765
3694
7410
8082
1432
7388
3694
19407
5768
228515
33667
11082
3425
6455
7361
26176
33768
2512
36839
100492
84005
364330
14822
22203
3665
10381
7393
3693
Control
of the
media
extract
9726
2310
5585
4772
6753
628
12950
4762
18484
92929
8481
3173
1537
3554
2129
1581
Table 4 Comparison of HPLC chromatogram peaks areas for analysis the
effect of addition of the S. cerevisiae yeast extract elicitor on M. macroura
Miq 509 hairy root cultures
Nevertheless, compounds L (M3), O, and U on the sample were
produced lower (more or less: 1/2 times, 1/3 times, and 1/3 times)
than on the control. Furthermore, the addition of the elicitor
inhibited a compound production, namely compound B which
was only found on the control of methanol extract. Thus it were
indicated that the addition of the elicitor was decreased some
secondary metabolites productions.
All compounds on the sample of media possessed concentrations
higher than on the control of media (see table 4). Even, the
compounds which possessed concentrations higher on the control
of extract methanol, compounds O and U, were released into the
sample of media with concentrations higher (more or less: 2 times
and 1.75 times) than into the control of media. Other compounds
which were released into the samplel of media with
concentrations higher than on the control of media, were
compounds C, E, F, G, H, I, J, K, N, P, Q, S, and compound T
(more or less: 1.3 times, 1.3 times, 5 times, 5 times, 4 times, 3
times, 21 times, 4 times, 6 times, 7 times, 2 times, 2 times, 1.5
times). Furthermore, there was a compound which only released
into the samplel of media, namely compound R. Nevertheless,
compound D which was not found on the control of methanol
extract, and compound U, were just released into the control of
media. All of these were indicated that the addition of S.
cerevisiae yeast extract elicitor was stimulated M. macroura Miq
509 hairy root cultures to release some compounds into media
with concentrations higher than their control, and to block release
of other compounds (compounds D and U).
According to bioactivity assay, the sample of the methanol extract
showed weak activity against human ovarian cancer cell line
(OVCAR-3) with IC50 values 49 μg/ml, and the control of the
methanol extract showed inactive against OVCAR-3 with IC50
values 102 μg/ml, thus the addition of the elicitor increased
activity against OVCAR-3. Chalcomoracin-M2, M4 and M7
showed activity against OVCAR-3 (IC50: 11; 11; and 12.57
μg/ml). The productions of these compounds were increased by
the elicitor addition. If the results of bioactivity assay connected with
the increase some compounds productions, then it can be suggested
that the addition of the elicitor of S. cerevisiae yeast extract on M.
macroura Miq 509 hairy root cultures was increased productions of
compounds which possesses activity against OVCAR-3.
OH
OH
HO
O
OH
O
O
OH
HO
Mulberrofuran P (M1)
Conclusions
From 35 g of the hairy root of M. macroura Miq 509 with the
addition of the Saccharomyces cerevisiae yeast extracts elicitor,
yielded four Diel Alder type adduct compounds, namely M1
(mulberrofuran P; 29 mg), M2 (chalcomoracin; 193 mg), M4 (20
mg), M7 (5 mg), an aliphatic compound, namely M3 (Intan 8; 3
mg); and two unknown aromatic compounds [M5 (9 mg); M6 (5
mg)]. M2, M4 and M7 were showed potent anticancer activity
against human ovarian cancer cell lines (OVCAR-3) (IC50: 11; 11;
and 12 μg/ml). Based on HPLC analysis and bioactivity assay
toward sample of the methanol extract and control of the
methanol extract, the addition of the S. cerevisiae yeast extract
elicitor on M. macroura Miq 509 hairy root cultures caused
increase of production of secondary metabolites which possess
activity against OVCAR-3.
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HO
OH
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HO
OH
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O
HO
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O
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OH
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22''
21''
4
3a
5
6
B
3
A
HO
HO
2'
2
7a O
1'
C
7
6'
3'
4'
11"
10"
OH
12''
13"
E
9''
OH
O
14"
20'' 19''
8''
OH
4''
F
5''
5'
3''
OH
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18''
15''
D
2''
6''
1"
16''
17''
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HO
7"
M4
24''
13''
22''
17''
20''
16'' O
15''
H
5''
HO
10''
O
8''
H
4''
4'
3''
3'
6''
2''
OH
1''
7''
M7
OH
2
8
1'
2'
H
3
4
OH 6'
5'
9''
19''
21''
11''
14''
HO 18''
25''
23''
OH
12''
5
7
1
6
Nelson, Ricky (2004), Isolasi Metabolit Sekunder Dari Akar Rambu Morus
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