Biooxidation Of Berberine By The Endophytic Fungus Coelomycetes Afkr-1 Isolated From Kayu Kuning [Archangelisia Flava (L.) Merr.: Menispermaceae].
BIOOXIDATION OF BERBERINE BY THE ENDOPHYTIC FUNGUS
COELOMYCETES AFKR-1 ISOLATED FROM KAYU KUNING
[Archangelisia flava (L.) MERR.: Menispermaceae]
ANDRIA AGUSTA1, YULIASRI JAMAL, PRAPTIWI AND AHMAD FATHONI
Laboratorium Bioscience, Botany Division, Research Center for Biology,
The Indonesian Institute of Sciences. Jl. Raya Bogor Km. 46, Cibinong 16911 West Java
Alamat korespondensi, E-mail: bislunatin@yahoo.com
ABSTRACT
Totaly eighteen kinds of endophytic fungi have been isolated from the young
stems of kayu kuning [Archangelisia flava (L.) Merr.] collected from Bogor Botanic
Garden. All of isolated fungi have been screened for their capability to transform the
host plant alkaloid berberine in two kinds of liquid mediums, potato dextrose broth
(PDB) and glucose-yeast extract-peptone (GYP) respectively. Biotransformation
reactions were monitored and analyzed by a normal phase thin layer chromatography
technique. The screening process revealed that the fungus Coelomycetes AFKR-1 could
be able to transform berberine into a biotransformed product in PDB medium. Scalingup biotransformation reactions (5 x 200 ml medium in 500 ml size Erlenmeyer) which
were incubated at room temperature with agitation at 120 rpm yielded 20 % conversion
rate of substrate into a product in 48 hrs. The chemical structure of biotransformed
product was proposed as a 7-N-oxide-berberine based on their spectroscopic data.
Keywords: Archangelisia flava, endophytic fungi, Coelomycetes AFKR-1, berberine,
biotransformation.
INTRODUCTION
Microorganisms have been paid attention by many researchers since they
possess a peculiar metabolic capacity, and shows an increasing demand on chemical and
pharmaceutical companies. The Merck Research Laboratory, USA screened more than
500 kinds of microbes for their capability to transform anticancer drugs, taxol and
cephalomannine in order to find out more water soluble derivatives (Chen et al., 2001).
From those biotransformation process, two hidroxy taxol derivatives and one hidroxy
cephalomannine derivative were achieved. The use of microbes was also widely applied
to produce an interesting derivatives from many kinds of steroidal drugs (Faramarzi et
al., 2008). Endophytic fungus Xylaria sp. isolated from the plant Cinchona pubescence
(Rubiaceae) reported can transform Cinchona alkaloids (quinine, quinidine, cinchonine,
cinchonidine) into 1-N-oxide derivatives and showing an anti-malarial activity with very
weak toxicity compared with the typical Cinchona alkaloid quinine (Shibuya et al.,
2003). Endophytic fungus Diaporthe sp. isolated from a tea plant Camelia sinensis also
reported can biotransform tea catechins into C-4 hydorxy derivatives, and could not
react with non tea catechins (Agusta et al., 2005). Recently, the endophytic fungi
isolated from Thitonia diversifolia, Viguiera arenaria and Viguiera robusta showed
their capability to transform a neuroleptic drug, thioridazine through regio- and
stereoselective reactions (Borges et al., 2008).
MATERIALS AND METHODS
Plant Materials
The youg stems of kayu kuning [Archangelisia flava (L.) Merr.] were collected
from Bogor Botanical Garden in 2007 and identified at Herbarium Bogoriensis, Botany
Division, Research center for Biology, Indonsian Institute of Sciences.
Isolation of Endophytic Fungi
Young stems of kayu kuning were washed by tap water and then cut intopieces
with the length of 1 cm, followed by sterilization by soaking the stems in the 70%
alcohol for 2 minutes, then soaked in the NaOCl for 5 minutes and soaked again in 70%
alcohol for 30 second. The sterilized stems were sliced with sterile knife and then put on
Corn Meal Malt Agar (CMMC) media that had been added with 0.05 mg/ml
chloramphenicol followed by incubation in the room temperature for 1 week. Every
colony of endophitic fungi were serially transfered onto Potato Dextrose Agar (PDA)
media untill pure colony were obtained. The obtained endophytic fungi were preserve in
-80 oC at LIPI-MC.
Screening for Biotransformation of Berberine
Each of isolated endophytic fungi were cultivated in 20 ml of two kinds of liquid
medium (in 50 ml size Erlenmeyer), Potato dextrose broth (PDB) and glucose-yest
extract-peptone (GYP). After incubated at room temperature (26 – 28 oC) on a
reciprocal shaker (100 rpm)for 5 days, 2 ml (1 mg/ml in methanol) of berberine was
added into the medium, and con
t inue incubate on identical conditions. The
biotransformation reactions were monitored every days (until one week) throught a
series of thin layer chromatography (TLC) analysis of reaction extracts on a TLC plate
GF254 (Merck) with developing sovent CHCl3 – MeOH – HAc ( 6: 1 : 1 drop). The
TLC chromatogram patterns were visualized under expossure of UV light at 254 and
355 nm, and dragendorf reagent.
Biotransformation of Berberine by the Endophytic Fungus Coelomycetes AFKR-1
The endophytic fungus AFKR-1 was inoculated into the glucose–yeast extract–
peptone medium (200 ml) and cultivated for 5 d under shaking at 90 rpm at 26-28 °C. A
solution of berberine (20 mg) in MeOH (20 ml) was added to the cultivation medium
and shaking continued for 1 d. The reaction mixture was filtrated to remove the fungus
bodies. The filtrate was extracted with EtOAc and concentrated under reduced pressure
to give a product 2( 5 mg), which was purified by Sephadex LH-20 column
chromatography (MeOH) and a preparative TLC (silica gel 60 F254, CHCl3-MeOH,
3:1) to afford a biotransformed product (20 mg, 20%).
The chemical structure of biotransformed product was deduce based on their
spectroscopic data [NMR (500 MHz) and TOF-MS data].
RESULTS AND DISCUSSION
Eighteen kinds of endophytic fungi isolates from young stems of kayu kuning
plant [Arcghangelisia flava (L.) Merr.] collected in Bogor Botanical Garden were
obtained in our laboratory. Through a set of screening test, it was found one of
endophytic filamentous fungus Coelomycetes AFKR-1 could biotransform berberine
into a transformed product in GYP medium at 26 – 28 oC and 120 rpm. Scaling-up
reaction to be 1 L (5 x 200 ml in 500 ml size Erlenmeyer) with addition 100 mg (20 mg
each) of substrate berberine could achieved 20 mg (20 %) of a biotransformed product
(Fig. 1).
The 1H-NMR and
13
C-NMR spectra of the biotransformed product showed an
identical signal patterns to those of berberine (Tabel 1.). The above 1D-NMR spectra
revealed there is no change in chemical environment of proton and carbon atoms in
berberine molecule and NMR analysis could not resolve the chemical structure of the
biotransformed product. On the other hand, both of biotransformed product and
berberine gave a different spots on a TLC plate (Silica gel GF254, Merck) and different
peaks on an HPLC analysis (reverse phase C18 HPLC, 90 % acetonitrile, 40 oC, 340
nm). Furthermore, the biotransformed product was subjected into a high resolution
TOF-MS analysis. The TOF-MS spectrum of biotransformed product shows the ion
peak at [M+] 353, 16 amu. bigger than the ion peak of berberine (Fig 2.). These result
revealed that the biotransformation reaction of berberine by the endophytic fungus
Coelomycetes AFKR-1 could placed one additional oxygen atom (mass 16 amu.) into
the berberine molecule. Based on the above data, we assume that chemical structure of
the biotransformed product is a new berberine 7-N-oxide derivative (submitted for
publication). In addition, the biological activity of biotransformed product berberine 7N-oxide is in under progress.
The biotransformation reaction of berberine into 7-N-oxide derivative is quite
similar with biotransformation reaction of Cinchona alkaloids by endophytic fungus
Xylaria sp. which was also reported produce N-oxide derivatives (Shibuya et al., 2003).
O
O
N
O
Coelomycetes AFKR-1
O
O
CH 3
CH 3
O
N
O
O
26 %
O
berberine
CH 3
CH 3
berberine 7-N-oxide
Fig 1.
Tabel 1. 13C-NMR and 1H-NMR data for biotransformed product and berberine in DMSO-d6.
No.
Atoms
Biotransformed Product
13
C-NMR
1
H-NMR
7.80 (1H, s)
Berberine
13
C-NMR
105.4
1
105.4
2
147.7
147.7
3
149.8
149.8
4
108.4
4a
130.7
7.09 (1H, s)
108.4
130.7
1
H-NMR
7.80 (1H, s)
7.09 (1H, s)
5
26.3
3.20 (2H, dd, J=6.2 Hz)
26.3
3.20 (2H, dd, J=6.2
6
55.2
4.93 (2H, dd, J=6.2 Hz)
55.2
Hz)
8
145.5
9.90 (1H, s)
145.5
4.93 (2H, dd, J=6.2
8a
121.4
121.4
Hz)
9
143.6
143.6
9.90 (1H, s)
10
150.4
150.4
11
126.7
8.20 (1H, d, J=9.1 Hz)
126.7
12
123.5
8.01 (1H, d, J=9.1 Hz)
123.6
12a
133.0
13
120.2
13a
133.0
8.20 (1H, d, J=9.1
120.2
Hz)
137.5
137.5
8.01 (1H, d, J=9.1
13b
120.5
120.5
Hz)
9-OCH3
61.9
4.10 (3H, s)
61.9
10-OCH3
57.0
4.07 (3H, s)
57.1
O-CH2-O
102.1
6.17 (2H, s)
102.1
8.94 (1H, s)
8.95 (1H, s)
4.09 (3H, s)
4.07 (3H, s)
6.17 (2H, s)
Fig. 2.
Acknowledgement
This works were partially supported by IFS grant (No. F/4613-1) and LIPI
Internal research fund (DIPA).
LITERATURE CITED
Agusta, A., S. Maehara, K. Ohashi, P. Simanjuntak and H. Shibuya, 2005, Stereo
selective oxidation at C-4 of flavans by the endophytic fungus Diaporthe sp.
isolated from a tea plant, Chem. Pharm. Bull., 53 (12), 1565-1569.
Borges KB., WDS Borges, MT Pupo, PS Bonato. 2008. Stereoselective analysis of
thioridazine-2-sulfoxide and thioridazine-5-sulfoxide: An investigation of racthioridazine biotransformation by some endophytic fungi, J. Pharm. Biomed.
Anal., 46, 945-952
Chen TS., Xiaoh Li, Dan Bollag, Yeuh-Chuen Liu and Ching-jer Chang. 2001.
Biotransformation of taxol. Tetrahedron Letters, 42, 3787-3789.
Faramarzi MA., M Aghelnekad, MT Yadzi, M Amini, N Hajarolasvadi. 2008.
Metabolism of andros-4-en-3,17-dione by filamentous fungus Neurospora
crassa, Steroids, 73, 13-18.
Ping Xu, Dongliang Hua, and Cuiqing Ma, 2007, Microbial transformation of
propenylbenzenes for natural flavour production. TRENDS in Biotechnology, 25,
571-576.
Shibuya H., C Kitamura, S Maehara, M Nagahata, H Winarno, P Simanjuntak, HS Kim,
Y Wataya and K Ohashi. 2003. Transformation of Chincona alkaloids into 1-Noxide derivatives by endophytic Xylaria sp. isolated from Chincona pubescence.
Chem. Pharm. Bull., 51, 71-74.
COELOMYCETES AFKR-1 ISOLATED FROM KAYU KUNING
[Archangelisia flava (L.) MERR.: Menispermaceae]
ANDRIA AGUSTA1, YULIASRI JAMAL, PRAPTIWI AND AHMAD FATHONI
Laboratorium Bioscience, Botany Division, Research Center for Biology,
The Indonesian Institute of Sciences. Jl. Raya Bogor Km. 46, Cibinong 16911 West Java
Alamat korespondensi, E-mail: bislunatin@yahoo.com
ABSTRACT
Totaly eighteen kinds of endophytic fungi have been isolated from the young
stems of kayu kuning [Archangelisia flava (L.) Merr.] collected from Bogor Botanic
Garden. All of isolated fungi have been screened for their capability to transform the
host plant alkaloid berberine in two kinds of liquid mediums, potato dextrose broth
(PDB) and glucose-yeast extract-peptone (GYP) respectively. Biotransformation
reactions were monitored and analyzed by a normal phase thin layer chromatography
technique. The screening process revealed that the fungus Coelomycetes AFKR-1 could
be able to transform berberine into a biotransformed product in PDB medium. Scalingup biotransformation reactions (5 x 200 ml medium in 500 ml size Erlenmeyer) which
were incubated at room temperature with agitation at 120 rpm yielded 20 % conversion
rate of substrate into a product in 48 hrs. The chemical structure of biotransformed
product was proposed as a 7-N-oxide-berberine based on their spectroscopic data.
Keywords: Archangelisia flava, endophytic fungi, Coelomycetes AFKR-1, berberine,
biotransformation.
INTRODUCTION
Microorganisms have been paid attention by many researchers since they
possess a peculiar metabolic capacity, and shows an increasing demand on chemical and
pharmaceutical companies. The Merck Research Laboratory, USA screened more than
500 kinds of microbes for their capability to transform anticancer drugs, taxol and
cephalomannine in order to find out more water soluble derivatives (Chen et al., 2001).
From those biotransformation process, two hidroxy taxol derivatives and one hidroxy
cephalomannine derivative were achieved. The use of microbes was also widely applied
to produce an interesting derivatives from many kinds of steroidal drugs (Faramarzi et
al., 2008). Endophytic fungus Xylaria sp. isolated from the plant Cinchona pubescence
(Rubiaceae) reported can transform Cinchona alkaloids (quinine, quinidine, cinchonine,
cinchonidine) into 1-N-oxide derivatives and showing an anti-malarial activity with very
weak toxicity compared with the typical Cinchona alkaloid quinine (Shibuya et al.,
2003). Endophytic fungus Diaporthe sp. isolated from a tea plant Camelia sinensis also
reported can biotransform tea catechins into C-4 hydorxy derivatives, and could not
react with non tea catechins (Agusta et al., 2005). Recently, the endophytic fungi
isolated from Thitonia diversifolia, Viguiera arenaria and Viguiera robusta showed
their capability to transform a neuroleptic drug, thioridazine through regio- and
stereoselective reactions (Borges et al., 2008).
MATERIALS AND METHODS
Plant Materials
The youg stems of kayu kuning [Archangelisia flava (L.) Merr.] were collected
from Bogor Botanical Garden in 2007 and identified at Herbarium Bogoriensis, Botany
Division, Research center for Biology, Indonsian Institute of Sciences.
Isolation of Endophytic Fungi
Young stems of kayu kuning were washed by tap water and then cut intopieces
with the length of 1 cm, followed by sterilization by soaking the stems in the 70%
alcohol for 2 minutes, then soaked in the NaOCl for 5 minutes and soaked again in 70%
alcohol for 30 second. The sterilized stems were sliced with sterile knife and then put on
Corn Meal Malt Agar (CMMC) media that had been added with 0.05 mg/ml
chloramphenicol followed by incubation in the room temperature for 1 week. Every
colony of endophitic fungi were serially transfered onto Potato Dextrose Agar (PDA)
media untill pure colony were obtained. The obtained endophytic fungi were preserve in
-80 oC at LIPI-MC.
Screening for Biotransformation of Berberine
Each of isolated endophytic fungi were cultivated in 20 ml of two kinds of liquid
medium (in 50 ml size Erlenmeyer), Potato dextrose broth (PDB) and glucose-yest
extract-peptone (GYP). After incubated at room temperature (26 – 28 oC) on a
reciprocal shaker (100 rpm)for 5 days, 2 ml (1 mg/ml in methanol) of berberine was
added into the medium, and con
t inue incubate on identical conditions. The
biotransformation reactions were monitored every days (until one week) throught a
series of thin layer chromatography (TLC) analysis of reaction extracts on a TLC plate
GF254 (Merck) with developing sovent CHCl3 – MeOH – HAc ( 6: 1 : 1 drop). The
TLC chromatogram patterns were visualized under expossure of UV light at 254 and
355 nm, and dragendorf reagent.
Biotransformation of Berberine by the Endophytic Fungus Coelomycetes AFKR-1
The endophytic fungus AFKR-1 was inoculated into the glucose–yeast extract–
peptone medium (200 ml) and cultivated for 5 d under shaking at 90 rpm at 26-28 °C. A
solution of berberine (20 mg) in MeOH (20 ml) was added to the cultivation medium
and shaking continued for 1 d. The reaction mixture was filtrated to remove the fungus
bodies. The filtrate was extracted with EtOAc and concentrated under reduced pressure
to give a product 2( 5 mg), which was purified by Sephadex LH-20 column
chromatography (MeOH) and a preparative TLC (silica gel 60 F254, CHCl3-MeOH,
3:1) to afford a biotransformed product (20 mg, 20%).
The chemical structure of biotransformed product was deduce based on their
spectroscopic data [NMR (500 MHz) and TOF-MS data].
RESULTS AND DISCUSSION
Eighteen kinds of endophytic fungi isolates from young stems of kayu kuning
plant [Arcghangelisia flava (L.) Merr.] collected in Bogor Botanical Garden were
obtained in our laboratory. Through a set of screening test, it was found one of
endophytic filamentous fungus Coelomycetes AFKR-1 could biotransform berberine
into a transformed product in GYP medium at 26 – 28 oC and 120 rpm. Scaling-up
reaction to be 1 L (5 x 200 ml in 500 ml size Erlenmeyer) with addition 100 mg (20 mg
each) of substrate berberine could achieved 20 mg (20 %) of a biotransformed product
(Fig. 1).
The 1H-NMR and
13
C-NMR spectra of the biotransformed product showed an
identical signal patterns to those of berberine (Tabel 1.). The above 1D-NMR spectra
revealed there is no change in chemical environment of proton and carbon atoms in
berberine molecule and NMR analysis could not resolve the chemical structure of the
biotransformed product. On the other hand, both of biotransformed product and
berberine gave a different spots on a TLC plate (Silica gel GF254, Merck) and different
peaks on an HPLC analysis (reverse phase C18 HPLC, 90 % acetonitrile, 40 oC, 340
nm). Furthermore, the biotransformed product was subjected into a high resolution
TOF-MS analysis. The TOF-MS spectrum of biotransformed product shows the ion
peak at [M+] 353, 16 amu. bigger than the ion peak of berberine (Fig 2.). These result
revealed that the biotransformation reaction of berberine by the endophytic fungus
Coelomycetes AFKR-1 could placed one additional oxygen atom (mass 16 amu.) into
the berberine molecule. Based on the above data, we assume that chemical structure of
the biotransformed product is a new berberine 7-N-oxide derivative (submitted for
publication). In addition, the biological activity of biotransformed product berberine 7N-oxide is in under progress.
The biotransformation reaction of berberine into 7-N-oxide derivative is quite
similar with biotransformation reaction of Cinchona alkaloids by endophytic fungus
Xylaria sp. which was also reported produce N-oxide derivatives (Shibuya et al., 2003).
O
O
N
O
Coelomycetes AFKR-1
O
O
CH 3
CH 3
O
N
O
O
26 %
O
berberine
CH 3
CH 3
berberine 7-N-oxide
Fig 1.
Tabel 1. 13C-NMR and 1H-NMR data for biotransformed product and berberine in DMSO-d6.
No.
Atoms
Biotransformed Product
13
C-NMR
1
H-NMR
7.80 (1H, s)
Berberine
13
C-NMR
105.4
1
105.4
2
147.7
147.7
3
149.8
149.8
4
108.4
4a
130.7
7.09 (1H, s)
108.4
130.7
1
H-NMR
7.80 (1H, s)
7.09 (1H, s)
5
26.3
3.20 (2H, dd, J=6.2 Hz)
26.3
3.20 (2H, dd, J=6.2
6
55.2
4.93 (2H, dd, J=6.2 Hz)
55.2
Hz)
8
145.5
9.90 (1H, s)
145.5
4.93 (2H, dd, J=6.2
8a
121.4
121.4
Hz)
9
143.6
143.6
9.90 (1H, s)
10
150.4
150.4
11
126.7
8.20 (1H, d, J=9.1 Hz)
126.7
12
123.5
8.01 (1H, d, J=9.1 Hz)
123.6
12a
133.0
13
120.2
13a
133.0
8.20 (1H, d, J=9.1
120.2
Hz)
137.5
137.5
8.01 (1H, d, J=9.1
13b
120.5
120.5
Hz)
9-OCH3
61.9
4.10 (3H, s)
61.9
10-OCH3
57.0
4.07 (3H, s)
57.1
O-CH2-O
102.1
6.17 (2H, s)
102.1
8.94 (1H, s)
8.95 (1H, s)
4.09 (3H, s)
4.07 (3H, s)
6.17 (2H, s)
Fig. 2.
Acknowledgement
This works were partially supported by IFS grant (No. F/4613-1) and LIPI
Internal research fund (DIPA).
LITERATURE CITED
Agusta, A., S. Maehara, K. Ohashi, P. Simanjuntak and H. Shibuya, 2005, Stereo
selective oxidation at C-4 of flavans by the endophytic fungus Diaporthe sp.
isolated from a tea plant, Chem. Pharm. Bull., 53 (12), 1565-1569.
Borges KB., WDS Borges, MT Pupo, PS Bonato. 2008. Stereoselective analysis of
thioridazine-2-sulfoxide and thioridazine-5-sulfoxide: An investigation of racthioridazine biotransformation by some endophytic fungi, J. Pharm. Biomed.
Anal., 46, 945-952
Chen TS., Xiaoh Li, Dan Bollag, Yeuh-Chuen Liu and Ching-jer Chang. 2001.
Biotransformation of taxol. Tetrahedron Letters, 42, 3787-3789.
Faramarzi MA., M Aghelnekad, MT Yadzi, M Amini, N Hajarolasvadi. 2008.
Metabolism of andros-4-en-3,17-dione by filamentous fungus Neurospora
crassa, Steroids, 73, 13-18.
Ping Xu, Dongliang Hua, and Cuiqing Ma, 2007, Microbial transformation of
propenylbenzenes for natural flavour production. TRENDS in Biotechnology, 25,
571-576.
Shibuya H., C Kitamura, S Maehara, M Nagahata, H Winarno, P Simanjuntak, HS Kim,
Y Wataya and K Ohashi. 2003. Transformation of Chincona alkaloids into 1-Noxide derivatives by endophytic Xylaria sp. isolated from Chincona pubescence.
Chem. Pharm. Bull., 51, 71-74.