Synthesis of a new allelopathic agent from the biotransformation of ent-15α-hydroxy-16-kauren-19-oic acid with Fusarium proliferatum

Article abstract of DOI:10.1080/14786419.2017.1290614

The use of kaurane diterpenes as substrates in fungal biotransformation to achieve bioactive compounds has been widely reported. In this work, the natural product kaurenoic acid, a diterpene widely distributed in the plant Kingdom, was chemically converted into ent-15α-hydroxy-kaur-16-en-19-oic acid (1). Substrate 1 was subjected to biotransformation by the fungus Fusarium proliferatum, furnishing a new derivative, ent-2α,15α-dihydroxy-kaur-16-en-19-oic acid (2). The structure of metabolite 2 was deduced on the basis of spectroscopy and MS data. Derivative 2 showed allelopathic activity on germination and growth of root and stem of lettuce (Lactuca sativa), inhibiting 100% of germination and growth of roots and stem, at higher concentration assayed (10^?4?mol/L).

Full text of DOI:10.1080/14786419.2017.1290614

Natural Product Research
Formerly Natural Product Letters
ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: http://www.tandfonline.com/loi/gnpl20
Synthesis of a new allelopathic agent from
the biotransformation of ent-15α-hydroxy-16-
kauren-19-oic acid with Fusarium proliferatum
A. D. Rocha, H. da S. Vieira, J. A. Takahashi & M. A. D. Boaventura
To cite this article: A. D. Rocha, H. da S. Vieira, J. A. Takahashi & M. A. D. Boaventura (2017):
Synthesis of a new allelopathic agent from the biotransformation of ent-15α-hydroxy-16-kauren-19-
oic acid with Fusarium proliferatum, Natural Product Research
To link to this article: http://dx.doi.org/10.1080/14786419.2017.1290614
View supplementary material
Published online: 28 Feb 2017.
Submit your article to this journal
Article views: 14
View related articles
View Crossmark data
Full Terms & Conditions of access and use can be found at
http://www.tandfonline.com/action/journalInformation?journalCode=gnpl20
Download by: [The UC San Diego Library]
Date: 11 May 2017, At: 13:52

Natural Product research, 2017
ꢁꢁp://ꢂx.ꢂꢃi.ꢃꢄg/10.1080/14786419.2017.1290614
ꢀ
Synthesis of a new allelopathic agent from the
biotransformation of ent-15α-hydroxy-16-kauren-19-oic acid
with Fusarium proliferatum
A. D. Rocha, H. da S. Vieira, J. A. Takahashi and M. A. D. Boaventura
dꢅpꢆꢄꢁꢆmꢅnꢁꢃ ꢂꢅ Qꢇímiꢈꢆ, univꢅꢄꢉiꢂꢆꢂꢅ Fꢅꢂꢅꢄꢆꢊ ꢂꢅ Minꢆꢉ Gꢅꢄꢆiꢉ, Bꢅꢊꢃ hꢃꢄizꢃnꢁꢅ, Bꢄꢆziꢊ
ABSTRACT
ARTICLE HISTORY
rꢅꢈꢅivꢅꢂ 25 Jꢇꢊy 2016
aꢈꢈꢅpꢁꢅꢂ 6 Jꢆnꢇꢆꢄy 2017
Theuseofkauranediterpenesassubstratesinfungalbiotransformation
to achieve bioactive compounds has been widely reported. In
this work, the natural product kaurenoic acid, a diterpene widely
distributed in the plant Kingdom, was chemically converted into ent-
KEYWORDS
Biꢃꢁꢄꢆnꢉfꢃꢄmꢆꢁiꢃn; Fusarium
proliferatum; kꢆꢇꢄꢆnꢅ
ꢂiꢁꢅꢄpꢅnꢅꢉ; ꢆꢊꢊꢅꢊꢃpꢆꢁꢀiꢈ
ꢆꢈꢁiviꢁy; Lactuca sativa
1
5α-hydroxy-kaur-16-en-19-oic acid (1). Substrate 1 was subjected to
biotransformation by the fungus Fusarium proliferatum, furnishing a
new derivative, ent-2α,15α-dihydroxy-kaur-16-en-19-oic acid (2). The
structure of metabolite 2 was deduced on the basis of spectroscopy
and MS data. Derivative 2 showed allelopathic activity on germination
and growth of root and stem of lettuce (Lactuca sativa), inhibiting
1
00% of germination and growth of roots and stem, at higher
−
4
concentration assayed (10 mol/L).
1
. Introduction
Biotransformation of less polar natural products abundant in nature is an interesting strategy
for obtaining more polar hydroxylated derivatives for biological screenings (Mutafova et al.
2016). Functionalisation of kaurane diterpenes is of special interest since biologically active
compounds of this class frequently bear hydroxyl groups in the skeleton (Cheenpracha
et al. 2006; Chavan et al. 2011; Rico-Martínez et al. 2014; Ruan et al. 2014; Wu et al. 2014;
Hegazy et al. 2015;Tian et al. 2015; Mutafova et al. 2016). Hydroxylation of kaurane diterpenes
by fungal biotransformation has been widely reported (Fraga et al. 2013; Hegazy et al. 2015)
by a variety of filamentous fungi such as Cephalosporium aphidicola (Boaventura et al. 1994),
CONTACT M. a. d. Bꢃꢆvꢅnꢁꢇꢄꢆ
mꢆꢄiꢆmꢅꢊiꢆ@qꢇi.ꢇfmg.bꢄ
sꢇppꢊꢅmꢅnꢁꢆꢊ ꢂꢆꢁꢆ fꢃꢄ ꢁꢀiꢉ ꢆꢄꢁiꢈꢊꢅ ꢈꢆn bꢅ ꢆꢈꢈꢅꢉꢉꢅꢂ ꢆꢁ ꢀꢁꢁp://ꢂx.ꢂꢃi.ꢃꢄg/10.1080/14786419.2017.1290614.
©
2017 Infꢃꢄmꢆ uK limiꢁꢅꢂ, ꢁꢄꢆꢂing ꢆꢉ tꢆyꢊꢃꢄ & Fꢄꢆnꢈiꢉ Gꢄꢃꢇp

2
A. D. ROCHA ET AL.
Mucor plumbeus (Fraga et al. 2010), Beauveria sulfurescens (Furtado et al. 2013) and some
Rhizopus species such as R. nigricans (Garcia-Granados et al. 1990), R. oligosporus (Taveepanich
et al. 2010) and R. stolonifer (Boaventura et al. 1995).
The most commonly occurring biotransformation in kaurane diterpenes is the
hydroxylation and some positions are more frequently activated than others (Takahashi
et al. 2014). Despite the usefulness of biotransformation in this field, a great challenge is to
find micro-organisms able to consistently introduce a hydroxyl group with regioselectivity
in order to develop bench or industrial processes on a rational form.
Fungi from Fusarium genus are a good tool for biotransformation of several classes of
natural products. F. fujikuroi has successfully converted 15α-hydroxy-ent-kaur-9(11)-16-dien-
19-oic acid into its 3β,6β-dihydroxy derivative after an incubation period of 6 days (Fraga
et al. 2013). F. oxysporum, isolated as an endophyte from the plant Catharanthus roseus, was
able to convert vinblastine into vincristine (Kumar, Patil, et al. 2013; Kumar, Rai, et al. 2013;
Kumar 2016). F. oxysporum has also been reported to promote catalytic ring-D lactonisation
using several steroids besides its inability to promote hydroxylation in several positions of
steroid skeleton (Peart et al. 2011; Zhang et al. 2013). Fusarium proliferatum is reported to
produce hydrolases involved in the biosynthesis of important antitumour agents (Su et al.
2006). In kaurane biotransformation field, this fungal species is able to selectively hydroxylate
kaurane diterpenes at C-2, a position rarely reported to be affected by biotransformation
(
Rocha et al. 2010).
The aim of this study was to carry on the biotransformation of ent-15α-hydroxy-16-
caur-9(11)-dien-19-oic (1) by the filamentous fungus F. proliferatum in order to synthesise
derivatives with improved allelopathic activity.
2
. Results and discussion
After 14 days of biotransformation with F. proliferatum, a product (2) was isolated as a solid
on a high yield (26.4%) (Figure 1). The IR spectrum revealed the presence of hydroxyl
−
1
−1
1
(
3346 cm ) and carboxyl (1695 cm ) groups. In the H NMR spectrum of 2 were observed
signals at δ 5.20 and δ 5.09, corresponding to methylene hydrogens at C-17 (δ 108.7),
H
H
C
showing that the exocyclic double bound was retained in the structure of the product. In
the same way, the signal of H-15 (δ 3.75) and the signals of both methyl groups H –18 and
H
3
H –20 (δ 1.28 and 0.95, respectively) were present in the spectrum.
3
H
F. proliferatum
1
2
Figure 1. cꢃnvꢅꢄꢉiꢃn ꢃf ꢈꢃmpꢃꢇnꢂ 1 inꢁꢃ 2 by F. proliferatum.

NATURAL PRODUCT RESEARCH
3
1
3
The C NMR and DEPT data (Table S1) indicated the presence of 20 carbons, including a
carboxyl (δ 180.4) and two oxygenated methine carbons (δ 82.7 and δ 64.1), indicating
C
C
C
a new hydroxyl group in the product. Analysis of HSQC correlation map showed correlation
between the carbon at δ 64.1 with the corresponding hydrogen signal at δ 4.05 (H-2). The
C
H
chemical shifts of C-1 and C-3 of the product were down fielded (δ 49.4 and 46.7, respec-
C
tively) in relation to the corresponding carbon atoms in the starting material (Table S1).
These data indicated that the new hydroxylation took place at C-2. In the COSY spectrum of
2
, it was possible to characterise the structural unity formed by H-1 (δ 2.22), H-2 (δ 4.05)
H H
2
and H-3 (δ 2.41). Key HMBC correlations of C-2 (δ 64.1) (J ) and H-1 (δ 0.7) and H-3 (δ 2.4
and δ 0.93) (J ), C-3 (δ 46.7) and H -18 (δ 1.28); C-1 (δ 49.4) and H -3 (δ 0.93); H -20 (δ
H
C
H
H
3
H
C
3
H
C
2
H
3
H
0
.95) and C-9 (δ 53.4) were used to establish the structure of 2.
C
The configuration of the hydroxyl group at C-2 (δ 64.1) was established using the corre-
C
lations observed in a NOESY experiment, where H-2 (δ 4.05) showed special interaction
H
with H -20 (δ 0.95), which is the α-face; therefore, H-2 is also in the α-face and, consequently,
3
H
the hydroxyl group at C-2 has β-orientation. Prefix ent- implies that the compounds belong
to ent-kaurane series and means complete inversion at all configuration stated in relation
to the fundamental parent structure, according to the official nomenclature rule for this class
of compounds (Giles 1999). It means that, if a kaurane diterpenes is drawn as a β-OH con-
figuration, the official nomenclature is ent-α-hydroxy (ent-α = β). The methylene hydrogen
atoms at H-15 did not present spatial correlation with H-9 (δ 1.07), but instead, they are
H
spatially close to H-13 (δ 2.73), H-14 equatorial (δ 1.44) and H-17 (δ 5.2). This is only pos-
H
H
H
sible for a 15β configuration. Complete NMR assignment for compounds 1 and 2 is presented
in Table S1.
+
High-resolution mass spectrum of 2 furnished a fragment [M + 1] (m/z 335.2459) as well
as fragments with m/z 317.2349 [M − H O] and m/z 3517.2349 [M − OH] . This is, by the
+
+
+
2
test of our knowledge, the first report on the isolation of compound 2.
The biotransformation of diterpene 1 by F. proliferatum into a kaurane hydroxylated in
carbon 2 (2) shows the regioselective consistency of this fungal species towards hydroxyl-
ation of kaurane diterpenes.
Hydroxylation of diterpenes at C-2 as a result of a fungal biotransformation has not been
frequently reported (Fraga et al. 2012;Takahashi et al. 2014) but has been previously reported
from F. proliferatum (Rocha et al. 2010). This coherent behaviour of this species towards
molecules with kaurane skeleton, together with the high initial biotransformation yield,
indicates a good perspective of a rational use of this fungal species for selective hydroxylation
of kaurane and structurally related molecules.
Since kaurane diterpenes have been extensively reported to influence plants growth,
compounds 1 and 2 were assayed for allelopathic activity. ent-2α,15α-dihydroxy-kaur-16-
en-19-oic acid (2) promoted inhibition of the stem and root growth in three concentrations
evaluated (Figure 2). At a concentration of 10−4 mol/L this compound completely blocked
growth of both, stems and roots of lettuce, when compared to the control. Compound 2
also blocked germination completely at the concentration of 10− mol/L. In the other con-
centrations evaluated, this compound exhibited only a weak inhibitory effect. Comparison
of the results exhibited by 1 and 2 showed, in overall, the biotransformation product pre-
sented stronger activity on Lactuca sativa germination.
4

4
A. D. ROCHA ET AL.
Figure 2. effꢅꢈꢁ ꢃf ꢈꢃmpꢃꢇnꢂ 2 ꢃn gꢅꢄminꢆꢁiꢃn ꢆnꢂ ꢄꢆꢂiꢈꢆꢊ ꢆnꢂ ꢉꢀꢃꢃꢁ ꢊꢅngꢁꢀ ꢃf Lactuca sativa.
Nꢃꢁꢅꢉ: Vꢆꢊꢇꢅꢉ ꢆꢄꢅ pꢄꢅꢉꢅnꢁꢅꢂ ꢆꢉ pꢅꢄꢈꢅnꢁꢆgꢅ ꢂiffꢅꢄꢅnꢈꢅꢉ fꢄꢃm ꢁꢀꢅ ꢈꢃnꢁꢄꢃꢊ, wiꢁꢀ zꢅꢄꢃ ꢄꢅpꢄꢅꢉꢅnꢁing ꢆn ꢃbꢉꢅꢄvꢅꢂ vꢆꢊꢇꢅ iꢂꢅnꢁiꢈꢆꢊ
ꢁꢃ ꢁꢀꢅ ꢈꢃnꢁꢄꢃꢊ, ꢆ pꢃꢉiꢁivꢅ vꢆꢊꢇꢅ ꢄꢅpꢄꢅꢉꢅnꢁing ꢉꢁimꢇꢊꢆꢁiꢃn, ꢆnꢂ ꢆ nꢅgꢆꢁivꢅ vꢆꢊꢇꢅ ꢄꢅpꢄꢅꢉꢅnꢁing inꢀibiꢁiꢃn.
3
. Experimental
3
.1. General
Column chromatography was performed on silica gel (Merck, Darmstadt, Germany) (230–400
mesh). Reagents for culture medium preparation were acquired from Labsynth and Biobrás
(Brazil) and Acumedia (Michigan, USA). Solvents used for chromatography were purchased
from Merck (Darmstadt, Germany). Deuterated solvents and reagents were purchased from
Aldrich Chemical Co. (Milwaukee, WI). Infrared spectra were recorded on a Spectrum One,
1
13
Perkin-Elmer spectrometer from Pharmacy Faculty, UFMG (Brazil). H and C NMR spectra, sub
1
1
1
1
1
13
1
13
spectra DEPT 135 and H- H COSY, H- H NOESY, H- C HMBC, and H- C HSQC experiments
were obtained on BRUKER AVANCE DPX200 and DRX400 spectrometers at the High Resolution
Magnetic Resonance Laboratory (LAREMAR) from the Chemistry Department of the Federal
University of Minas Gerais (MG, Brazil). Spectra were measured at 400 (100) or 200 (50) MHz at
3
00 K, and the spectrometers were equipped with inverse detection 5 mm multinuclear head
1
13
H/ C. The compound was dissolved in CDCl , and transferred to a 5 mm o.d. NMR tube.
3
Chemical shifts (δ) were registered in ppm and tetramethylsilane (TMS) was used as an internal
reference standard to chemical shift (δ 0). Bi-dimensional (2D) NMR spectra were acquired
under standard conditions. Data processing was carried out on SGI workstation using the Bruker
(DRX 400) software. HRMS (ESI) was performed by Dr. Adriano Pimenta from the Biological
Sciences Institute, UFMG, on a ESI Q-TOFMicro apparatus equipped with eletrostatic electrospray
ionisation source at 80°C, capillary voltage of 1600 V in methanol/TFA 0.1% in the positive mode.
3
.2. Substrate preparation
Kaurenoic acid was obtained from the ethanol extract of Wedelia paludosa aerial parts (Vieira
et al. 2001). To this compound (1.0 g; 3.31 mmol), selenium dioxide (0.46 g, 4.19 mmol) in
dioxane (30 mL; 352.7 mmol) was added. The mixture was heated under reflux in nitrogen
atmosphere for 2 h, at 110°C, under magnetic stirring. The residue (1.57 g) was submitted
to successive column chromatography in silica gel to furnish the product of the allylic

NATURAL PRODUCT RESEARCH
5
oxidation (1) as a dark yellow gum (503.5 mg; 50.4% yield). ent-15α-hydroxy-kaur-16-en-19-
−
1
oic acid (1): C H O (316 g/mol). IR (λ , cm ): 3473 (O–H); 2956–2936 (C–H); 1689 (C=O);
2
0
28
1
3
max
13
1
460–700 (C–O). H and C NMR (CDCl , δ) (Table S1).
3
3
.3. Biotransformation experiment
F. proliferatum (CML 287), donated by Dr. Ludwig H. Pfenning (Lavras Mycological Collection,
UFLA, MG, Brazil), was stored in PDA under refrigeration. This strain was reactivated and
added to two 500 mL conical flasks containing sterile liquid medium (200 mL/flask) with the
following composition (g/L): iron sulphate (0.01 g/L), magnesium sulphate (0.5 g/L), potas-
sium chloride (0.5 g/L), dibasic potassium phosphate (1.0 g/L), sodium nitrate (3.0 g/L) and
sucrose (30.0 g/L) in distilled water. The flasks were maintained under magnetic shaking, at
room temperature (25 ± 3°C) for four days. Portions of this material (15 mL each) were asep-
tically transferred to 24 conical flasks containing fresh sterile medium and incubated under
orbital stirring for 48 h. Then, the substrate 1 (0.36 g) in ethanol was evenly distributed into
the flasks (0.5 mL/flask). The biotransformation was carried out under orbital shaking
(
100 rpm). After 14 days, flasks contents were filtered (Whatman #1 filter paper) for mycelia
removal and the broth was extracted with ethylacetate. After solvent removal, the residue
4.28 g) was column chromatographed on silica gel (100 g) using an elution gradient of
hexane, CH Cl , EtOAc and MeOH. Unaltered starting material was recovered in the fractions
(
2
2
eluted from CH Cl :EtOAc (7:3) (30.4 mg, 8.44% of the amount added). A group of fractions
2
2
eluted with 100% EtOAc (1.27 g) further purified by silica gel column, from where a solid
eluted with CH Cl /ethyl acetate (1:1) was obtained. This compound showed to be pure by
2
2
TLC and was identified as ent-2α,15α-dihydroxy-kaur-16-en-19-oic acid (2).
ent-2α,15α-dihydroxy-kaur-16-en-19-oic acid (2). Bright yellow solid (95.1 mg, 26.4%
−
1
yield); MM: C H O (334 g/mol). mp 159–162°C. IR (λ , cm ): 3346 (O–H); 2930 (C–H); 1695
20
30
4
max
+
+
(
C = O); 1435–771 (C–O e C–C). HRMS: 335.2459 [M + 1]; 317.2349 [M − H O]; 3517.2349
2
+
+
1
13
[
M − OH] . H and C NMR (CDCl , δ) (Table S1).
3
3
.4. Biological assay
Compound 2 was evaluated on the growth of radical and shoot of L. sativa (lettuce) (cv Grand
Rapids) purchased from Isla Pak (Brazil), in triplicate according to methodology previously
described (Vieira et al. 2005; Freitas et al. 2015). Briefly, 25 seeds were placed in Petri dishes
−4
−6
containing a filter paper as a support (9.0 cm) with 10 mL of the test compound (10 , 10
−8
and 10 M) or control solution prepared with deionised water. pH values [buffered with
0 mM 2-(N-morpholino) ethanesulphonic acid, MES] were adjusted to 6.0–6.5 with NaOH
1
solution. Dishes were covered with Parafilm to reduce evaporation and incubated in the dark
at 25°C, in a controlled-environment growth chamber, for 5 days. After this time, number of
germinated seeds were counted (a seed was considered as germinated when the radicle was
at least 0.2 mm long) and lengths of radical and shoots were measured. During measurement,
dishes were kept at 4°C to avoid subsequent growth. The effects on growth are given as per
cent differences from control and consist of the differences (in cm) between mean values of
seed with tested compound and mean value for control (seeds without addition of test
compound)/mean values for control × 100. Thus, zero represents the control, positive values
represent stimulation of the studied parameter and negative value represents inhibition.

6
A. D. ROCHA ET AL.
Acknowledgements
The authors thank Brazilian Funding Agencies Conselho Nacional de Desenvolvimento Científico e
Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and
Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) for grants, scholarships and
financial support. Prof. Adriano Pimenta (ICB, UFMG) is fully acknowledged for obtaining the HRMS
spectra. Prof. Ludwig Pfennig (UFLA) is acknowledged for strain donation.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This work was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
[grant number 478853/2013-0], Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
and Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG)
ORCID
J. A. Takahashi
http://orcid.org/0000-0002-8831-1609
References
Boaventura MAD, Hanson JR, Hitchcock PB, Takahashi JA. 1994. The bio-transformation of ent-19-
hydroxykaur-16-en-15-one by Cephalosporium aphidicola. Phytochemistry. 37:387–389.
Boaventura MAD, Oliveira AB, Hanson JR, Hitchcock PB, Takahashi JA. 1995. The biotransformation of
methyl ent-15-oxokaur-16-en-19-oate by Rhizopus stolonifer and Mucor plumbeus. Phytochemistry.
40:1667–1669.
Chavan MJ, Wakte PS, Shinde DB. 2011. Analgesic and anti-inflammatory activities of 18-acetoxy-ent-
kaur-16-ene from Annona squamosa L. bark. Inflammopharm. 19:111–115.
Cheenpracha S, Yodsaoue O, Karalai C, Ponglimanont C, Subhadhirasakul S, Tewtrakul S, Kanjana-
opas A. 2006. Potential anti-allergic ent-kaurene diterpenes from the bark of Suregada multiflora.
Phytochemistry. 67:2630–2634.
Fraga BM, de Alfonso I, Gonzalez-Vallejo V, Guillermo R. 2010. Microbial transformation of two
1
5α-hydroxy-ent-kaur-16-ene diterpenes by Mucor plumbeus. Tetrahedron. 66:227–234.
Fraga BM, Bressa C, González-Vallejo V, González P, Guillermo R. 2012. Biotransformation of ent-kaur-
6-ene and ent-trachylobane 7β-acetoxy derivatives by the fungus Gibberella fujikuroi (Fusarium
1
fujikuroi). Phytochemistry. 81:60–70.
Fraga BM, González-Vallejo V, Guillermo R, Amaro-Luis JM. 2013. Microbiological transformation of two
1
8
5α-hydroxy-ent-kaur-9(11),16-diene derivatives by the fungus Fusarium fujikuroi. Phytochemistry.
9:39–46.
Freitas LB, Boaventura MAD, Santos WL, Stehmann JR, Junior DD, Lopes MT, Magalhães TF, da Silva DL,
de Resende MA. 2015. Allelopathic, cytotoxic and antifungic activities of new dihydrophenanthrenes
and other constituents of leaves and roots extracts of Banisteriopsis anisandra (Malpighiaceae).
Phytochem Lett. 12:9–16.
Furtado RA, Gunaherath GM, Bastos JK, Gunatilak AA. 2013. Microbial biotransformation of 16-alpha,
1
8
7-epoxy-ent-kaurane-19-oic acid by Beauveria sulfurescens ATCC 7159-F. Nat Prod Commun.
:1041–1044.
Garcia-Granados A, Martinez A, Ortiz A, Onorato ME, Arias JM. 1990. Microbial transformation of
tetracyclic diterpenes: conversion of ent-kaurenones by Curvularia and Rhizopus strains. J Nat Prod.
53:441–450.

NATURAL PRODUCT RESEARCH
7
Giles PM. 1999. Revised section F: Natural products and related compounds. Pure Appl Chem. 71:587–
43.
6
Hegazy MEF, Mohamed TA, ElShamy AI, Mohamed AEHH, Mahalel UA, Reda EH, Shaheen AM, Tawfik
WA, Shahat AA, Shams KA, Abdel-Azim NS, Hammouda FM. 2015. Microbial biotransformation as
a tool for drug development based on natural products from mevalonic acid pathway: a review. J
Adv Res. 6:17–33.
Kumar A. 2016. Vincristine and vinblastine: a review. Intern J Med Pharm Sci. 6:23–30.
Kumar A, Patil D, Rajamohanan PR, Ahmad A. 2013. Isolation, purification and characterization of
vinblastine and vincristine from endophytic fungus Fusarium oxysporum isolated from Catharanthus
roseus. PLoS One. 8:e71805. doi:10.1371/journal.pone.0071805.
Kumar V, Rai S, Gaur P, Fatima T. 2013. Endophytic fungi: novel sources of anticancer molecules. In:
Verma VC, Gange AC, editors. Advances in Endophytic Research. New Delhi: Springer; p. 389–422.
Rico-Martínez M, Medina FG, Marrero JG, Osegueda-Robles S. 2014. Biotransformation of diterpenes.
RSC Adv. 4:10627–10647.
Mutafova B, Mutafov S, Fernandes P, Berkov S. 2016. Microbial transformations of plant origin
compounds as a step in preparation of highly valuable pharmaceuticals. J Drug Metab Toxicol.
7:204. Available from: http://dx.doi.org/10.4172/2157-7609.1000204
Peart PC, McCook KP, Russell FA, Reynolds WF, Reese PB. 2011. Hydroxylation of steroids by Fusarium
oxysporum, Exophiala jeanselmei and Ceratocystis paradoxa. Steroids. 76:1317–1330.
Rocha AD, Santos GCD, Fernandes NG, Pfenning LH, Takahashi JA, Boaventura MAD. 2010. Hydroxylation
at carbon-2 of ent-16-Oxo-17-norkauran-19-oic acid by Fusarium proliferatum. J Nat Prod. 73:1431–
1433.
RuanY, Ma BJ, Li LZ, Wang XL. 2014. Specific 12α-hydroxylation of grandiflorenic acid by permeabilised
fungus Fusarium graminearum. Nat Prod Res. 28:677–679.
Su JH, Xu JH, Lu WY, Lin GQ. 2006. Enzymatic transformation of ginsenoside Rg3 to Rh2 using newly
isolated Fusarium proliferatum ECU2042. J Mol Catal B: Enzym. 38:113–118.
Takahashi JA, Gomes DC, Lyra FH, dos Santos GF, Martins LR. 2014. The remarkable structural diversity
achieved in ent-kaurane diterpenes by fungal biotransformations. Molecules. 19:1856–1886.
Taveepanich S, Muangsin N, Saithong S, Pakawatchai C, Chaichit N, Roengsumran S, Petsom A. 2010.
Biotransformation of ent-kaur-16-en-19-oic acid by Absidia blakesleeana and Rhizopus oligosporus.
Nat Prod Rep. 24:1050–1058.
Tian T, JinY, MaY, Xie W, Xu H, Zhang K, DuY. 2015. Identification of metabolites of oridonin in rats with
a single run on UPLC-Triple-TOF-MS/MS system based on multiple mass defect filter data acquisition
and multiple data processing techniques. J Chrom B. 1006:80–92.
Vieira HS, Takahashi JA, Boaventura MAD. 2001. Constituents from aerial parts of Wedelia paludosa.
Fitoterapia. 72:854–856.
Vieira HS, Takahashi JA, Pimenta LPS, Boaventura DMA. 2005. Effects of kaurane diterpene derivatives
on germination and growth of Lactuca sativa seedlings. Zeit für Naturf. 60:72–78.
Wu H-Y, Zhan R, Wang W-G, Jiang H-Y, Du X, Li X-N, Li Y, Pu J-X, Sun H-D. 2014. Cytotoxic ent-kaurane
diterpenoids from Isodon wikstroemioides. J Nat Prod. 77:931–941.
Zhang H, Ren J, Wang Y, Sheng C, Wu Q, Diao A, Zhu D. 2013. Effective multi-step functional
biotransformations of steroids by a newly isolated Fusarium oxysporum SC1301. Tetrahedron.
69:184–189.