Biotransformation of extracted digitoxin from Digitalis lanata by Streptomyces

Article abstract of DOI:10.1691/ph.2011.0859

The biotransformation of digitoxin and some of its derivatives extracted from Digitalis lanata by Streptomyces isolated species was investigated. Cultures of a Streptomyces strain designated EUSA-2003B, isolated from an Egyptian soil sample, efficiently induced selective 12β-hydroxylation of the steroid aglycone of digitoxin (DT) and its α-acetyl and β-methyl derivatives. The transformation reaction was performed within a 5-day fermentation process, products were isolated and their aglycone moiety was obtained by acid hydrolysis and their structures were elucidated by ¹³C and ¹H NMR. The biotransformation resulted mainly digoxin (DG,～87%), meanwhile, digoxigenone (DGON,～7.0%)was also afforded as a side product. The present study revealed that: 1-Streptomyces isolate EUSA2003B harbors its specific 12β-hydroxlase and has the capability to transform DT and it's α-acetyl and α-methyl derivatives into their corresponding digoxins at reasonable yields. 2-The minor structural differences in the trisaccharide side chain seemed ineffective on the transformational capability of this organism. 3-The Streptomyces might also possess a specific glycosidase that splits the saccharidic side chain beside another dehydrogenase that oxidizes C₃ at the steroid nucleus into its ketone form (DGON).

Full text of DOI:10.1691/ph.2011.0859

ORIGINAL ARTICLES
1
2
Chemistry Department , Faculty of Science , King Khalid University Abha, The Kingdom of Saudi Arabia; Biochemistry
Department, Faculty of Science, Ain Shams University, Abbasiya, Cairo, Egypt
Biotransformation of extracted digitoxin from Digitalis lanata by
Streptomyces
1
2
2
2
S. Keshk , M. Mostafa , F. Tawfik , I. Elshemy
Received November 19, 2010, accepted December 23, 2010
Prof. Sherif Khesk, Chemistry Department, King Khalid University, P. O. Box 9004, Abha, The Kingdom of Saudi
Arabia
sherifkeshk@yahoo.com
Pharmazie 66: 458–462 (2011)
doi: 10.1691/ph.2011.0859
The biotransformation of digitoxin and some of its derivatives extracted from Digitalis lanata by
Streptomyces isolated species was investigated. Cultures of a Streptomyces strain designated EUSA-
2003B, isolated from an Egyptian soil sample, efficiently induced selective 12␤-hydroxylation of the steroid
aglycone of digitoxin (DT) and its ␣-acetyl and ␤-methyl derivatives. The transformation reaction was per-
formed within a 5-day fermentation process, products were isolated and their aglycone moiety was obtained
13
1
by acid hydrolysis and their structures were elucidated by C and H NMR. The biotransformation resulted
mainly digoxin (DG, ∼87%), meanwhile, digoxigenone (DGON, ∼7.0%) was also afforded as a side product.
The present study revealed that: 1-Streptomyces isolate EUSA2003B harbors its specific 12␤-hydroxlase
and has the capability to transform DT and it’s ␣-acetyl and ␤-methyl derivatives into their corresponding
digoxins at reasonable yields. 2-The minor structural differences in the trisaccharide side chain seemed
ineffective on the transformational capability of this organism. 3-The Streptomyces might also possess a
specific glycosidase that splits the saccharidic side chain beside another dehydrogenase that oxidizes C3
at the steroid nucleus into its ketone form (DGON).
1
. Introduction
steran moiety (aglycone nucleus) in different positions including
␤ (13); 5␤ (14); 7␤ and 12 ␤ (Szelecky et al. 1980). However,
1
Phytochemical reviews pointed out that cell cultures of Digitalis
lanata(foxglove)synthesizeandtransformvariablecardioactive
substances known as digitalis cardenolides. Cardenolides are
divided into 6 series (A to F), based on their “genin” part. Series
A glycosides, like lanatoside A and digitoxin are the most abun-
dant members, whereas type C, like digoxin are those clinically
used (Kreis et al. 1990). Digoxin, is still used in the treatment of
early (Albrecht et al. 1982) and recent trials failed to achieve
reasonable success using fungi for 12␤-hydroxylation (Pádua
et al. 2007). This study, as an alternative solution, is aiming to
exploit cultures of a Streptomyces isolates for reproducible bio-
transformation of relatively toxic type “A” cardioactive drugs
into their corresponding therapeutically desired forms (C type)
via 12-␤ hydroxylation enzyme. Table 1
+
+
congestive heart failure by reducing the Na /K ATPase activ-
ity and increasing, consequently, the cardiac muscle contractility
(
Matthew et al. 1974). Chemically, transformations of DTs into
2
. Investigations, result and discussion
DGs have been tried in many laboratories using known strate-
gies, but unfortunately, reported fastidious and time-consuming
Much interest was paid to the microbial transformation of toxic
cardioactive drugs to their relatively less or non-toxic deriva-
tives of high therapeutic index using cell cultures of plants
(Albrecht et al. 1982). The low therapeutic index of DT (Tanaka
et al. 1990) which made DG clinically preferable has moti-
vated several researchers to find a way of transforming DT into
DG with sustaining the pharmacologic activity and high yield
under mild fermentation conditions (Volkov 1994).Therefore,
microbial biotransformation of DT-containing wastes, under
optimized conditions, into their corresponding DGs is still a
reasonable approach to achieve this goal.
Members of the cardiac glycosides are either transformed into
cardioactive derivatives not known to occur in nature (Padua
et al. 2005), or to valuable products in high yields (Pérez-Alonso
et al. 2009). Therefore, application of microorganisms to induce
stereo-specific structural modifications of cardenolides is still a
promising approach for economic production of commercially
required drugs. In fact, a few microbial species, mostly fila-
mentous fungi, showed remarkable activity to hydroxylate the
(Kreis et al. 1990), bacteria (Fuska et al. 1987), Streptomyces
(Baljet 1968) and fungi (Pádua et al. 2007). Filamentous fungi
and Streptomyces present rates of biomass growth higher than
those of plant cell cultures, what turns out their transforma-
tions more feasible for large scale applications. Nonetheless,
little work was devoted to the of use soil Streptomyces iso-
lates to perform this task. This prompted us to exploit soil
Streptomyces for achieving the biotransformation of digitox-
ins into digoxins. After being subjected to transformation, DT
and its ␣-acetyl and ␤-methyl derivatives, two compounds
were obtained from the growing cultures of the Streptomyces
EUSA2003B, namely digoxigenin and digoxigenone (Table 2),
while the untransformed portions were detected as digitoxi-
genin. Digitoxin and its ␣-acetyl and ␤-methyl derivatives were
4
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ORIGINAL ARTICLES
Table 1: Chemical structures of the cardenolides under investigation
Cardenolides
Abbreviation
R1
R2
Digitoxin
Digoxin
DT
DG
␣ -AcDT
DTG
␤ -MeDT
DGG
H
OH
H
H
H
Dox- ␤1,4- Dox- ␤1,4- Dox- ␤1-
Dox- ␤1,4- Dox- ␤1,4- Dox- ␤1-
␣ -AcDox- ␤1,4- Dox- ␤1,4- Dox- ␤1-
H
␤-Me-Dox- ␤1,4- Dox- ␤1,4- Dox- ␤1-
H
␣
-Acetyldigitoxin
Digitoxigenin
-Methyldigitoxin
Digoxigenin
␤
OH
Dox = digitoxose; ␣ –AcDox = ␣ -Acetyldigitoxose; ␤- MeDox = ␤ Methyldigitoxose
Table 2: Yield and retention times (R
cardenolides
t
) of the assessed
taken by the transforming Streptomyces. Besides digoxigenin
(DGG), another 3-keto-12 ␤-hydroxy aglycone (Digoxigenone,
DGON) was evolved since their proton chemical shifts were
consistent with its structure (Table 3). The elemental analysis of
extracted digitoxin is showed 64.37% carbon, 8.43% hydrogen,
and 27.19% oxygen. While elemental analysis of biotrans-
formed digoxin is showed 63.06% carbon, 8.62% hydrogen, and
Cardenolides
Abbreviation
Yield# and Rt(min)
Digitoxin
DT
(24.7)
Digoxin
DG
∼ 87% (22.3)
Digitoxigenin
Digoxigenin
Digoxigenone
DTG
DGG
DGON
(24.0)
28.69%oxygen. Bothdigitoxinanddigoxingivebluegreenfluo-
∼5.0% (25.9)
7.0% (19.4)
rescence after spraying with acidic ferric chloride reagent. Then
when exposed to UV light digitoxin gives orange fluorescence
and digoxin gives blue fluorescence (Fig. 1). Concerning the
physical properties, the ultraviolet spectrum showed one band
at ␭ max 220 nm for biotransformed digoxin which satisfies the
#Based on 10 mg cardinolide added to 2-day old growing culture of Streptomyces EUSA2003B
allowed to grow for further 5 consecutive days
␭
max of standard digoxin. On the other hand digitoxin gives
employed as substrates for biotransformation by the Strepto-
myces isolate after being individually added to 48 h growing
cultures. Their transformation products were isolated, purified
and acid-hydrolyzed liberating their modified aglycones to be
quantified by HPLC (Table 1). Their purity and authenticity
were comparatively attested by spectroscopic techniques. The
transformation medium components lacking the organism (C-2
culture) were found not to affect the added cardenolides, since
no transformation products were detected after the same period
one band at ␭ max 240 nm. Mass spectroscopy analysis showed
molecular ion peak at 766, 781 and 782 for digitoxin, digoxin
and biotransformed digoxin respectively which represent the
molecular weight for the digitoxin, digoxin and bio transformed
1
digoxin (Figs. 2–4). From the H NMR spectrum of digoxigenin
a complex profile and the only signals easily assigned were those
of H-3␣(␦ 4.14), H-21(␦ 4.90 and ␦ 4.80) and H-22 (␦ 5.94)
which confirmed the presence of C3-OH group and the ␣ and
␤-unsaturated lactone ring. Singlets at ␦ 0.81 and ␦ 0.97 were
Table 3: 13C and H NMR assignments of digitoxigenin and the biotransformation products by Streptomyces EUSA-2003B
1
Chemical shifts
Digitoxigenin
(␦/ppm)
13C
(1H)
Digoxigenin
Digoxigenone
1
2
3
4
1␣;1␤
2 ␣;2 ␤
29.6(1.49) (1.49)
27.9(1.53) (1.53)
29.6(1.49) (1.49)
27.9 (1.53) (1.53)
37.8 (2.11) (1.45)
37.9(2.53) (2.11)
3 ␣
66.8(4.13)
66.7(4.14)
215.7(–)
4
5 ␤
6 ␣;6 ␤
7 ␣;7 ␤
8 ␤
␣;4 ␤
33.3(1.34) (1.89)
36.0(1.78)
26.5(1.87) (1.30)
21.2(1.25) (1.71)
41.8(1.56)
33.3(1.39) (1.94)
36.0(1.84)
26.3(1.90) (1.32)
21.6(1.26) (1.71)
41.4(1.54)
43.0(2.78) (1.95)
45.4(1.81)
27.8(1.93) (1.34)
22.4(1.31) (1.84)
42.1(1.66)
34.3(1.91)
36.3
5
6
7
8
9
9␣
35.5(1.62)
35.4
32.4(1.66)
35.2
10
11
12
13
14
15
16
17
18
19
20
21
22
23
11␣;11␤
12␣;12␤
21.3(1.46) (1.46)
40.0(1.40) (1.53)
49.6
30.4(1.68) (1.27)
75.1(3.38) (–)
55.5
30.8(1.65) (1.34)
75.4(3.47) (–)
57.3
85.5
85.9
86.6
15␣;15␤
16␣;16␤
17␣
33.1(2.13) (1.71)
26.9(1.87) (2.16)
51.0(2.79)
15.7(0.88)
23.7(0.96)
174.5(5.00)
73.4 (4.81)
117.6(5.87)
174.4
33.3(1.89) (1.82)
27.4(1.67) (2.16)
45.6(3.33)
8.9 (0.81)
23.6(0.97)
174.2(4.90)
73.6(4.81)
117.8(5.94)
174.5
33.5(2.07) (1.77)
28.4(1.95) (2.15)
47.0(3.35)
9.9 (0.81)
22.8(1.05)
177.3(4.99)
75.4(4.91)
117.8(5.99)
178.4
#
Values between brackets refer to chemical shifts of hydrogen.
Pharmazie 66 (2011)
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ORIGINAL ARTICLES
Fig. 1: TLC of digitoxin, digoxin and biotransformed digoxin
readily assigned indicating the presence of C18 and C19 methyl
groups (Table 3). The hydroxylation position was unambigu-
ously defined on the basis of the chemical shift changes recorded
for digoxigenin (the acid-hydrolysis end product of transformed
DT,␣-acetyl and ␤-Me DT as well) which allowed to allocate
1
2 ␤-OH group. These results revealed that the bio transformed
Fig. 3: Mass spectroscopy of digoxin
digoxin is identical to the authentic digoxin. Microbial transfor-
mation of digitoxin and some of its derivatives was successfully
studied using Streptomyces sp., however, use of Fusarium cil-
iatum (Pádua et al. 2005) led to negative result which was
hypothetically attributed to the insufficient impermeability of
fungal cells to the DT as compared to vegetal cell membranes.
In the present work, shortening of the reaction period to 5 days
afforded reasonable yields of the 12␤-hydroxylated products
(DG, DGG, and DGON) which is explainable on the basis of
the time-course production of these products (Fig. 5). A gradual
consumption of the fortified cardenolides started on 20 h after
addition to the 2-day old cultures accompanying corresponding
increase in their yields until complete consumption at the end of
incubation term. Digoxin and digoxigenone seem to be the final
Fig. 2: Mass spectroscopy of digitoxin
Fig. 4: Mass spectroscopy of biotransformed digoxin
4
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ORIGINAL ARTICLES
1
1
20
00
80
60
40
20
0
DT
DGG
DGON
0
20 40 60 80 100 120 140 160 180
Time (h)
Fig. 6: Electron micrograph of the selected Streptomyces strain BS-3 (X 10000)
(a)
1
20
cessful transformation of digitoxins might partially be due to
the use of cyclodextrin in the cultivation medium to increase
permeability, and hence, facilitate its penetration through the
cell walls of the Streptomyces. Studies of characterization, bio-
chemical and physiological properties of selected Streptomyces
strain EUSA-2003B revealed that morphologically, the spores
of selected Streptomyces EUSA-2003B are tubular with spiky
surface, and showing branching. Cultivation of selected Strepto-
myces strain on starch nitrate agar, glucose-nitrate, nutrient-agar
and potato-agar media leads to the observation of variations
in pigment secreted, aerial and substrate mycelia. The isolated
Streptomyces EUSA-2003B possessed strongly coagulates and
peptonizes skimmed milk and reduce nitrate, which indicate
that Streptomyces EUSA-2003B possessed strong proteolytic
and reducing activities. On the other hand, Streptomyces EUSA-
100
8
6
4
2
0
0
0
0
0
DT
DGG
DGON
0
20 40 60 80 100 120 140 160 180
Time (h)
2003B could moderately liquefy gelatin and failed decompose
cellulose (Fig. 6).
(b)
The current study has demonstrated the capability of Strepto-
myces EUSA2003B to convert DT, ␣-acetyl and ß-methyl-DT
into their corresponding 12ß-hydroxy derivatives in a 5-days fer-
mentation process. The final yield (87%) of the therapeutically
needed digoxin is reasonably high which gives this strain the
advantage to be commercially feasible if submitted to further
biotechnological applications.
1
00
9
8
7
6
5
4
3
2
1
0
0
0
0
0
0
0
0
0
0
DT
DGG
DGON
3. Experimental
3.1. Instruments and chemicals
The UV absorption spectra were determined with PYE Unicon SP-800 and
IR spectra were carried out using PYE Unicon SP-400. Mass spectroscopy
was carried out with mass spectrophotometer at electron energy (70 mv)
◦
1
13
and final temperature 197 . The H NMR, C NMR, HMQC as well as
0
20 40 60 80 100 120 140 160 180
Time (h)
1H1H-COSY spectra were recorded using Bruker DRX-400 spectrometer
1
13
(
H400 MHz and C 100 MHz) using TMS and internal standard for H and
C nuclei. Chemical shift values (␦)are expressed in ppm while J coupling
values are given in Hertz (Hz). Digitoxin, digoxin, digitoxigenin, digoxi-
genin, ␣-acetyl and ␤–methyl derivatives of DT and DG were supplied by
Sigma Chemical co (USA). Solvents and other chemicals including those
for HPLC and NMR spectroscopic methods are of high chromatographic
grade (Aldrich, Human and Sigma Co.). Bi-distilled water was routinely
used for purifications and water-containing solvent mixtures.
(c)
Fig. 5: Biotransformation (%) vs time (h) of digitoxin (a), ␣-acetyl-digitoxin (b) and
␤-methyl-digitoxin (c) by Streptomyces EUSA2003B
transformation products evolved by 12␤-hydroxylase activity
harbored in the implicated organism (Table 3). The nature of
the glycosidic side chains seems of minor interference in the
yield of the bioconversion products since the productivity of
digoxigenin from ␣-acetyl (Fig. 5b) and ␤-methyl-digitoxins
3
.2. Cultivation of Digitalis lanata
Seeds of Digitalis lanata were purchased from European Authorized Agri-
culture Center, Germany. Seed were cultivated in October 2005. The seeds
started to germinate after 15 days from cultivation and reached the beginning
of the fruiting stage after 9 months. Then, the leaves were cut and rapidly
◦
dried in circulating hot air oven at 55–60 C.
(Fig. 5C) was a bit lower than that from DT (Fig. 5A). The suc-
Pharmazie 66 (2011)
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ORIGINAL ARTICLES
3
.3. Microorganisms
monitored by removing portions (5 ml) of broth, aseptically, at regular time
intervals (Table 2), extracted and acid hydrolyzed (only DT, ␣-Acetyl and
-Me derivatives). Aliquots of the extracted products were dissolved in 1 ml
of MeOH:CHCl3 (9:1, v/v) for the injection into Shimadzo HPLC apparatus
and their retention times (Rt, Table 2) were measured. The characterization
of the products was accomplished by comparing their Rt with authentic
samples.
Streptomyces strains were isolated in pure cultures from soil samples
collected from different localities in Egypt (Al-Areesh, Northern Cost,
and Cairo). Out of 153 isolates, a Streptomyces culture given the code
EUSA-2003B possessed biotransformation capability during the prelimi-
nary screening program.
␤
3
.4. Cultures and cultivation conditions
3
.9. Characterization of the selected streptomyces EUSA-2003B
Isolation and preservation of the Streptomyces isolates in pure cultures were
−
1
Numerous characterization of Streptomyces EUSA-2003B was carried out
according to the method of Waksman (1967) that including morphological,
cultural, biochemical characterization.
carried on starch-nitrate medium contained (g.L tap water): NaNO3 (2.0),
KH2PO4 (1.0), MgSO4.5H2O (0.5) and 20 g of solubilized starch were even-
tually added (Yukari et al. 1996). Difco agar (2.2 g %) was melted and added
whenever solid medium was needed and the pH was adjusted to 7.0 before
sterilization (Waksman and Lechivalier 1961). For the screening and bio-
transformation experiments, the cultivation medium (CM) contained (g/L):
yeast extract (3.5), soybean meal (5.0), KH2PO4 (5.0) and glucose (20.0)
Tween 80 (0.15%) was finally added (Pádua et al. 2007).
References
Albrecht K, Máté G, Láng
deoxycardenolids with Streptomyces purpurascens. Z Allg Mikrobiol 22:
31–435.
T (1982) Transformation of 12-
4
3
.5. Extraction of digitoxin from Digitalis lanata leaves
Baljet H (1968). From Mohamed Y: A pharmacognostical study of certain
Digitalis species newly introduced into Egypt., MSc. Thesis Pharmacog-
nosy Dept., Faculty of Pharmacy, Cairo University.
Extraction of digitoxin was carried out using Yukari et al. method (1996).
In which, 25 ml of 50%methanol was added to 250 mg of leaf powder after
ultrasonication for 1.5 h in an ice water bath the extract was filtered and then
was dissolved in 3 ml of methanol then in 50 ml of ethyl acetate. Thin layer
chromatographic (silica G254) analysis was used according to the Carvalhas
and Figueria method (1973) using chloroform-acetone-acetic acid (70: 30:
Carvalhas L, Figueira A (1973) Comparative study of thin-layer chro-
matographic techniques for separation of digoxin, digitoxin and their
metabolites. J Chromatogr 86: 254–260.
ˇ
Fuska J, Proksa B, Khandlová A, Sturdíková M (1987) Microbial
transformations of cardioglycosides. Appl Microbiol Biotechnol 26:
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semicontinuous production of deacetyllanatoside C in 20 litre airlift biore-
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0
.05) as eluting solvent. The method used for identification of digitoxin
and biotransformed digoxin was according to Matthew et al (1971). Where,
the mobile solvent methylene-methanol-formamide (80: 19: 1) and spray
reagent, acidic ferric chloride were used.
Padua R, Alaíde B, José, F Vieira J, Jacqueline T, Fernão J (2005) Bio-
transformation of digitoxigenin by Fusarium ciliatum Braz Chem Soc
3
.6. Biotransformation experiments
Erlenmeyer flasks (250 ml) containing 75 ml of sterile starch-nitrate
liquid medium were aseptically inoculated with fresh (7 day old culture)
suspension of the Streptomyces isolate. Cardenolides (10 mg) were
dissolved in DMF (1 ml) and added individually to 2-day growing cultures
of the Streptomyces followed by further incubation period of 5 days under
16: 614–616.
Pádua R, Oliveira A, Filho J, Takahashi J Silva M, Braga F (2007) Biotrans-
formation of digitoxigenin by Cochliobolus lunatus. J Braz Chem Soc 18:
1303–1310.
◦
Pérez-Alonso N, Wilken D, Annett G, Horst-Michael J, Kerns G, Capote-
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298–299.
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rotatory shaking (220 rpm) at 26–28 C. Cyclodextrin (0.2%) was added
to the DT-containing cultures to improve its permeability through the
cell membrane of the organism. Control experiments containing medium
plus Streptomyces isolate (C-1) and medium plus substrates (lacking the
organism, C-2) were run alongside the biotransformation experiments.
Immediately, at the end of incubation period (7 days), mycelia were
filtered off, the biotransformation products were sequentially extracted
with chloroform (3 × 50 ml) and chloroform/2-propanol (3:1,v/v) twice
(80 ml each) using separating funnel on a cotton swab, solvent was vacuum
◦
removed at 52 C, until residue. The obtained residues were combined,
acid hydrolyzed (see below) and submitted to preparative RP-TLC (21)
followed by preparative RP-HPLC (Kreis et al. 1990) where aqueous
acetonitrile/water (37: 63) was used for eluting the products.
Waksman S (1967) The Actinomycetes, classification, identification and
descriptions of Genera and Species. Baltimore: The Williams & Wilkins
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3
.7. Acid hydrolysis of biotransformation products
Volkov S (1994) Immunoassay of low-molecular-mass physiologically
active substances of plant and microbial origin encountered in plants.
Russ Chem Rev 63: 91–100.
Since the 12-␤-hydroxylation of the aglycone moiety of cardenolides under
investigation is the main target of the present study, we did not pay attention
to any probable changes that might occur in the trisaccharide side chain
(
␣-Methyl or ␤-acetyl-digitoxoses). Therefore, after extraction of the trans-
Yamauchi T, Abe F, Wan C (1987) Studies on Cerbera. V Minor Glycosides
of 17␣-Digitoxigenin from the Stems of Genus Cerbera. Chem Pharm
Bull 35: 4993–4995.
Yukari I, Youichi F, Megumi U, Tadahiko H, Mayumi M, Mitsuru Y (1996)
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by reversed phase thin-layer chromatography. J Chromatogr A 746:
◦
formation products, they were subjected to acid hydrolysis (1N HCl at 55 C
for 35 min) to perform easier RP-HPLC detection of the aglycone residue
of the transformed cardenolides.
3
.8. Time-course monitoring of biotransformation
255–260.
Under the abovementioned growth conditions, the capability of Strep-
tomyces EUSA-2003B to hydroxylate the fortified cardenolides was
4
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