New cardenolides from biotransformation of gitoxigenin by the endophytic fungus alternaria eureka 1e1bl1: Characterization and cytotoxic activities

molecules

Article

New Cardenolides from Biotransformation of Gitoxigenin by

the Endophytic Fungus Alternaria eureka 1E1BL1:

Characterization and Cytotoxic Activities

Erdal Bedir ^1,

* *

, Çig˘dem Karakoyun ^2,, Gamze Dog˘an ¹, Gülten Kuru ¹, Melis Küçüksolak ¹

and Hasan Yusufog˘lu ³

1

˙

Department of Bioengineering, Faculty of Engineering, Izmir Institute of Technology,

˙

35430 Urla-Izmir, Turkey; gamzedogan@iyte.edu.tr (G.D.); gultenkuru@iyte.edu.tr (G.K.);

meliskucuksolak@iyte.edu.tr (M.K.)

Department of Pharmacognosy, Faculty of Pharmacy, Ege University, 35100 Bornova-Izmir, Turkey

Department of Pharmacognosy, College of Pharmacy, Prince Sattam Bin Abdulaziz University,

Al-Kharj 11942, Saudi Arabia; h.yusufoglu@psau.edu.sa

2

3

˙

*

Correspondence: erdalbedir@iyte.edu.tr (E.B.); cigdem.karakoyun@ege.edu.tr (Ç.K.);

Tel.: +90-232-750-7389 (E.B.); +90-232-311-2598 (Ç.K.)

Abstract: Microbial biotransformation is an important tool in drug discovery and for metabolism

studies. To expand our bioactive natural product library via modiﬁcation and to identify possible

mammalian metabolites, a cytotoxic cardenolide (gitoxigenin) was biotransformed using the endo-

phytic fungus Alternaria eureka 1E1BL1. Initially, oleandrin was isolated from the dried leaves of

Nerium oleander L. and subjected to an acid-catalysed hydrolysis to obtain the substrate gitoxigenin

ꢀꢁꢂꢀꢃꢄꢅꢆꢇ

ꢀꢁꢂꢃꢄꢅꢆ

Citation: Bedir, E.; Karakoyun, Ç.;

Dog˘an, G.; Kuru, G.; Küçüksolak, M.;

Yusufog˘lu, H. New Cardenolides

from Biotransformation of

(yield; ~25%). After 21 days of incubation, ﬁve new cardenolides

1, 3, 4, 6, and 8 and three previously-

identiﬁed compounds and were isolated using chromatographic methods. Structural elucida-

2

,

5

7

Gitoxigenin by the Endophytic

Fungus Alternaria eureka 1E1BL1:

Characterization and Cytotoxic

Activities. Molecules 2021, 26, 3030.

https://doi.org/10.3390/

tions were accomplished through 1D/2D NMR, HR-ESI-MS and FT-IR analysis. A. eureka catalyzed

oxygenation, oxidation, epimerization and dimethyl acetal formation reactions on the substrate.

Cytotoxicity of the metabolites were evaluated using MTT cell viability method, whereas doxorubicin

and oleandrin were used as positive controls. Biotransformation products displayed less cytotoxicity

molecules26103030

than the substrate. The new metabolite

and 3.4 M against A549, PANC-1 and MIA PaCa-2 cells, respectively, without causing toxicity on

healthy cell lines (MRC-5 and HEK-293) up to concentration of 10 M. Our results suggest that A.

8 exhibited the highest activity with IC₅₀values of 8.25, 1.95

µ

Academic Editors: Horváth Györgyi,

µ

˙

Ihsan Çalıs¸ and Agnieszka

eureka is an effective biocatalyst for modifying cardenolide-type secondary metabolites.

Ludwiczuk

Keywords: Nerium oleander L.; cardenolides; oleandrin; gitoxigenin; biotransformation; endophytic

fungus; Alternaria eureka 1E1BL1; cytotoxicity

Received: 12 March 2021

Accepted: 12 May 2021

Published: 19 May 2021

Publisher’s Note: MDPI stays neutral

with regard to jurisdictional claims in

published maps and institutional afﬁl-

iations.

1. Introduction

Microbial biotransformation is one of the most preferred and powerful procedures

in natural product drug discovery and development studies enabling speciﬁc biochemi-

cal modiﬁcations of substrate molecules [1]. An enzyme, a living cell, or an inactivated

microorganism containing several speciﬁc enzymes can be used for biotransformation

processes leading to the production of new drug candidates with better properties. Mi-

crobial enzyme systems are able to catalyze regio- and stereo-speciﬁc reactions which

Copyright:

are challenging issues in chemical synthesis [2–4]. These enzymes are to employ diverse

Licensee MDPI, Basel, Switzerland.

This article is an open access article

distributed under the terms and

conditions of the Creative Commons

Attribution (CC BY) license (https://

creativecommons.org/licenses/by/

4.0/).

chemical reactions such as oxygenation, oxidation/reduction, cyclization, epoxidation,

dehydrogenation, acetylation/deacetylation and epimerization. Besides, most favored

metabolic products of the given substrate can be predicted especially via fungal biotrans-

formation [

5]. Thus, foreseeing metabolic fate of drug candidates provides insight into

mammalian drug metabolism at preclinical stages. Microbial transformation, as a modern,

Molecules 2021, 26, 3030. https://doi.org/10.3390/molecules26103030

https://ww w .mdpi.com/journal/molecules

Molecules 2021, 26, 3030

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time- and cost-efﬁcient molecular modiﬁcation approach, is of increasing interest to phar-

maceutical industries. Also, the use of microbial biotransformation has become one of the

essential parts of white biotechnology and green chemistry movements due to requiring

less energy and creating less waste [6].

Nerium oleander L. is a perennial evergreen shrub that belongs to the family Apocy-

naceae. It is a widely cultivated ornamental herb, native to the Mediterranean region. In

ethnobotanical literature, N. oleander is reported as a traditional medicine being used as

abortifacient and for the treatment of a wide range of diseases such as dermatitis, eczema,

skin cancer, viral infections, asthma, epilepsy, malaria, tumors, corns, leprosy, ringworm,

and warts [7]. All parts of this plant are known to be poisonous to human beings, animals,

and some insects. Both toxic effects and bioactivities of N. oleander are mainly attributed to

the cardiac glycoside content of the plant [8,9].

Recently, N. oleander has attracted a great deal of attention due to promising anticancer

activity of two patented water extracts: Anvirzel^®and Breastin^®11]. Anvirzel^®, a

[10,

sterile hot aqueous extract of N. oleander, has been recognized as a potent anticancer agent

against advanced solid tumors. A phase I clinical trial study demonstrated the safety of in-

tramuscular administration of Anvirzel^®up to the dose of 1.2 mL/m²/day. Other clinically

tested anticancer natural product, Breastin^®, is the cold-water extract of N. oleander exhibit-

ing selective antitumor activities against brain tumor SF268, lung carcinoma LXF1221L,

pancreas carcinoma PANC-1 and prostate cancer DU 145. Phytochemical studies have

demonstrated that potent anticancer activity of the water extract of N. oleander mostly arises

from its monoglycosidic cardenolide content such as oleandrin, odoroside A, odoroside

H, and neritaloside [11]. In particular, Anvirzel^®and Breastin^®contain considerably high

amounts of oleandrin, which is regarded as the principal active constituent anticancer [12].

Oleandrin, possesses a cardenolide-type aglycone called oleandrigenin together with

a dideoxy sugar moiety called oleandrose (Figure 1). The main physiological action of

cardenolides is cardiotonic effect and depends on disrupting sodium potassium pump

(Na⁺/K⁺-ATPase) functions leading to increased levels of intracellular calcium ions (Ca⁺⁺).

Thus, cardenolide-type compounds generate positive inotropic and negative chronotropic

action on heart muscles, and consistently improve the prognosis of patients with congestive

heart failure. In addition to anticancer and heart contractility promoting effects, oleandrin

is also known to possess anti-oxidant, diuretic, anti-inﬂammatory, neuroprotective and

anti-viral activities [13

potent anti-viral agent providing both prophylactic and curative treatment for COVID-19.

According to in vitro experiments, administration of oleandrin at 0.05 and 0.1 g/mL doses

–15]. Furthermore, a recent study has reported that oleandrin is a

µ

resulted in 78- and 100-fold reduction in infectious virus production, respectively [16].

Figure 1. Acid-catalysed hydrolysis of cardiac glycoside oleandrin and structures of oleandrin,

gitoxigenin and L-oleandrose (TFA: triﬂuoroacetic acid).

Gitoxigenin is desacetyl congener of oleandrigenin (Figure 1 ), of which the concen-

tration in biological samples of patients is commonly monitored as one of the follow-up

markers in cases of oleander poisoning [17]. Despite the fact that high concentrations of

Molecules 2021, 26, 3030

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gitoxigenin have poisonous effects, it has been known that certain doses have therapeutic

effects on congestive heart failure and atrial arrhythmia. Thus, gitoxigenin is a promising

natural product present in a variety of plants [18,19].

Based on our latest literature search, there are only a few studies on biological activity

of gitoxigenin [20

–

24]. Low et al. screened 502 natural products for anti-viral activity

40% inhi-

against dengue virus via immunoﬂuorescence assay with the hit criteria of

≥

bition, and gitoxigenin was detected as one of the 30 hit compounds exhibiting 49.01%

inhibition [25]. Xie et al. conducted a high throughput screening study on more than

3000 compounds and reported potent anticancer activity of gitoxigenin against human

colon cancer cell line [26]. Milutinovic et al. demonstrated the anticancer effect of gitoxi-

genin against HeLa cells through a homogeneous plate-reader assay (EC₅₀: 2.1 µM) [17].

Also, cytotoxic activity of gitoxigenin against three cancerous cell lines (human renal

adenocarcinoma, TK-10; human breast adenocarcinoma, MCF-7; and human melanoma,

UACC-62) was investigated via sulphorhodamine B colorimetric method in a previous

work by López-Lázaro et al., and gitoxigenin was reported as potent cytotoxic agent

showing IC₅₀values in the range of 0.415 to 2.83 µM [21].

Despite the promising potential of cardiac glycosides as drug candidates, their use in

medicinal applications is limited because of their aggressive pharmacokinetic and toxico-

logic behaviours. Thus, there is an urgent need for research on drug development studies

on cardenolide glycosides and their aglycones, in order to overcome these challenges. For

this purpose, biotransformation might be a useful tool for investigation of structure-activity

relationships (SAR), prediction of drug metabolism and discovery of new drug candi-

dates with increased bioavailability. However, only a few studies have been conducted on

microbial biotransformation of gitoxigenin [27,28].

Recently our group have performed molecular modiﬁcation studies on bioactive

natural products and reported interesting ﬁndings [29–32]. Taking into consideration of

these ﬁndings and our preliminary screening studies, A. eureka 1E1BL1 was proven to have

high transformation capacity on steroidal nuclei. Therefore, within the ﬁrst part of our

current work on the constituents of Nerium oleander L., the cardiac glycoside oleandrin was

transformed using the same endophytic fungus in addition to Phaeosphaeria sp. 1E4CS-1,

which resulted in isolation of ﬁve new oleandrin derivatives with signiﬁcant cytotoxic

activities [33].

In the present study, we aimed to perform a microbial biotransformation study on

gitoxigenin, which was obtained by acid-catalyzed hydrolysis of oleandrin. Incubation of

gitoxigenin with A. eureka 1E1BL1 has led to the production of ﬁve previously undescribed

cardenolides together with three known metabolites. Microbial oxygenations at C-7 and

C-12 sites were observed while dimethyl acetal formation on gitoxigenin core structure

was reported here for the ﬁrst time. All isolates and the substrate were evaluated for

cytotoxic activity against several cancer cell lines (PANC-1, MIA PaCa-2, DU 145 and A549)

and two healthy cell lines (MRC-5 and HEK-293) serving doxorubicin and oleandrin as

positive controls.

2. Results and Discussion

2.1. Isolation and Puriﬁcation of Oleandrin from N. oleander L. and Preparation of Substrate

Gitoxigenin and Obtaining Biotransformation Products

Oleandrin (4.0 g) was isolated from the dried leaves of N. oleander L. and subjected to

an acid-catalyzed hydrolysis affording gitoxigenin (25% yield) (Figure 1). The structural

identiﬁcation of gitoxigenin was established unambiguously by spectral methods (see

Section 3.6 and Table S1, in the Supplementary Materials). A twenty-one-day biotransfor-

mation experiment of gitoxigenin by Alternaria eureka 1E1BL1 yielded eight metabolites

(Figure 2).

Molecules 2021, 26, 3030

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Figure 2. Chemical structures of gitoxigenin (substrate) and eight biotransformation products

transformed by A. eureka 1E1BL1.

2.2. Structural Elucidations of Biotransformation Products

The ¹H and ¹³C NMR data of the new metabolites

Tables 1 and 2, respectively. Furthermore, the NMR data of

1

,

2

3

and

,

4

,

6

7

and

8 are presented in

, which were tentatively

identiﬁed in the previous studies, are herein reported for the ﬁrst time (Tables 1 and 2).

The molecular formula of was determined as C₂₃H₃₂O₆based on the major ion

1

peak at m/z 403.2123 [M-H]⁻(calcd. 403.2126 for C₂₃H₃₂O₆[M-H]⁻). The characteristic

hydroxymethine proton at C-3 was not present in the ¹H-NMR spectrum. Besides, the

downﬁeld shifts of C-2 and C-4 (ca. 8.3 ppm and 8.7 ppm) compared to those of gitoxigenin

implied an alteration on the ring A. Accordingly, in the ¹³C-NMR spectrum, the carbonyl

signal observed at δ_C212.7 was readily assigned to C-3, which was substantiated by the

observation of HMBC cross-peaks from C-3 (δ_C212.7) to one of H₂-2 (δ_H2.21, td, J = 14.4;

5.3 Hz) and H₂-4 (δ_H2.43, m) protons. Apart from the characteristic low-ﬁeld signals

observed, an additional oxymethine signal at δ_H3.84 was apparent, corresponding to a

carbon signal at δ_C69.1 in the HSQC spectrum. When the COSY spectrum was inspected

starting from the H₂-4 resonances (δ_H2.10 and 2.43), the spin system to the new resonance

at δ_H3.84 was tracked clearly [H₂-4/H-5 (δ_H1.86, m)/H₂-6 (δ_H1.50 and 1.83 m)/H-7 (δ_H

3.84, td, J = 10.6, 4.9 Hz)], suggested H-7 to be an oxymethine proton. The signiﬁcant

downﬁeld shifts of C-6 (δ_C35.9) and C-8 (δ_C45.6) (ca. 8.2 and 2.8 ppm compared to those

of gitoxigenin, respectively), and the long-distance correlations in the HMBC spectrum

from C-7 (δ 69.1) to H₂-6 (δ_H1.50 m; 1.83 m) and H-8 (δ_H1.69, dd, J=11.8; 10.4 Hz) veriﬁed

the monooxygenation at C-7 position (Figure 3).

The relative orientation of the hydroxy group was determined using 2D-NOESY.

The H-7 (δ_H3.84) signal showed NOESY correlation with the α-oriented H-9 (δ_H1.58 m)

establishing the β-orientation of the hydroxy group at C-7. Consequently, the structure of

compound 1 was characterized as 7β-hydroxy-3-oxogitoxigenin.

Molecules 2021, 26, 3030

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1

Table 1. H NMR data of gitoxigenin and compounds 1–8 (δ_H(ppm), (J, Hz)).

Position

1

Gitoxigenin

1.22 m; 1.27 m

1.24 m; 1.36 m

1

2

3

4

5

6

7

8

1.48 m; 2.05 dd

(14.2,4.5)

2.12 m; 2.47 td

(14.5, 5.1)

-

1.04 td (14.2,3.5);

1.82 m

1.06 d (14.5);

1.80 m

1.65 q (12.5);

1.96 m

1.11 td (14.2, 3.4);

1.85 m

1.41 m; 1.93 m

1.45 m; 2.00 m

1.25 m; 1.65 m

1.45 m; 2.11 m

2.12 m; 2.21 td

(14.4, 5.3)

-

2.20 d (16.1);

2.32 dd (14.5, 6.4)

-

2.12 m; 2.57 dd

(14.4, 5.5)

-

2

3

4

5

6

1.26 m; 1.77 m

-

1.37 m; 1.65 m

3.56 m

1.39 m; 1.69 m

3.59 m

3.82 t (2.8)

1.09 m; 1.67 td

(13.7, 3.2)

3.85 m

2.10 m; 2.43 m

1.86 m

2.06 m; 2.62 m

1.84 m

2.12 m; 2.66 t (14.3)

1.88 m

1.55 m; 1.77 m

1.67 m

1.49 m; 1.74 m

1.40 m

1.92 m; 1.99 m

1.97 m; 2.81 t (14.3)

1.82 m

1.53 m; 1.70 m

1.45 m

1.54 m

1.51 m

1.80 m; 2.14 td

(12.2, 4.9)

0.96 m; 1.63 m

1.50 m; 1.83 m

1.37 m; 1.90 m

1.58 m; 1.90 m

1.57 m; 1.92 m

1.35 m; 1.88 m

1.37 m; 1.91 m

1.38 m; 1.91 m

3.91 td (11.6, 11.2,

7

0.97 m; 1.57 m

3.84 td (10.6, 4.9)

1.24 m; 1.91 m

3.96 m

1.29 m; 1.84 m *

4.26 m

1.31 m; 1.91 m

1.31 m; 1.84 m

4.7)

8

9

1.34 m

1.37 m

1.69 dd (11.8, 10.4)

1.62 m

1.69 m

1.71 m

1.88 m

1.72 m

1.54 m

1.59 m

1.70 m

1.95 m

1.77 m

1.65 m

1.89 m

1.60 m

1.72 m

1.58 m

10

11

12

13

14

-

0.94 m; 1.11 m

1.11 m; 1.26 m

1.31 m; 1.37 m

1.25 m; 1.61 m

1.32 m; 1.46 m

1.35 m; 1.69 m

1.45 m; 1.63 m

3.36 dd (11.8, 4.1)

1.25 m; 1.41 m

1.25 m; 1.65 m

1.21 m; 1.42 m *

1.39 m; 1.55 m

1.25 m; 1.35 m

1.31 m; 1.49 m

1.30 m; 1.66 m

3.40 dd (11.8, 4.1)

1.30 m; 1.61 m

3.34 m

-

2.05 m; 2.40 dt

(14.4, 4.8)

4.44 brs

2.91 dd (7.0, 3.0)

0.96 s

1.49 dd (15.0, 2.4);

2.35 dd (14.9, 8.5)

4.43 td (8.3, 2.4)

2.88 d (7.9)

1.95 m; 2.36 dd

(14.2, 6.4)

4.41 t (6.5)

2.91 d (6.9)

1.90 m; 2.42 dd

(14.5, 6.4)

4.54 t (6.5)

2.96 d (7.1)

1.97 m; 2.59 dd

(14.5, 7.5)

4.58 m

3.58 d (7.6)

1.72 m; 2.63 dd

(14.9, 8.5)

4.66 td (8.2, 2.3)

3.14 d (7.9)

2.27 d (13.8); 2.67

1.79 m; 2.49 dd

(14.9, 7.6)

4.59 s

3.59 d (7.6)

1.82 m; 2.43 dd

(14.7, 7.6)

4.60 td (7.6, 1.7)

15

m

4.94 m

3.29 d (8.0)

1.11 s

0.93 s

-

16

17

18

19

20

3.59 m

0.87 s

0.97 s

-

0.66 s

-

0.90 s

0.98 s

-

0.98 s

1.02 s

-

0.89 s

1.06 s

-

0.92 s

0.93 s

-

0.89 s

1.06 s

-

0.97 s

-

4.96 d (18.4);

4.87 d (18.3);

5.02 d (18.3)

4.90 d (18.1);

5.05 d (18.1)

5.03 dd (18.5, 1.8);

5.14 dd (18.5, 1.9)

5.11 dd (18.4, 1.8);

5.17 dd (18.5, 1.8)

5.04 dd (18.3, 1.8);

5.18 dd (18.4, 1.9)

5.04 dd (18.5, 1.8);

5.19 dd (18.5, 1.9)

21

4.89 m; 5.06 m

5.57 m; 5.69 m

4.91 m

22

23

3-O-Me

5.71 s

5.88 s

5.97 s

6.01 s

5.97 s

-

3.12 s

3.19 s

5.94 s

6.25 s

6.03 s

-

CD₃OD/a drop of

CDCl₃/a drop of

CD₃OD

Solvent

CDCl₃

CD₃OD

CDCl₃

CD₃OD

Pyridine-d₅

CD₃OD

pyridine-d₅

* interchangeable.

Molecules 2021, 26, 3030

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Table 2. ¹³C-NMR data of gitoxigenin and compounds 1–8 δ_C(ppm).

Position

Gitoxigenin

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

22

23

30.7 t

28.5 t

67.6 d

34.1 t

37.3 d

27.7 t

22.4 t

42.8 d

36.5 d

36.3 s

22.0 t

40.8 t

51.3 s

85.5 s

43.8 t

73.0 d

59.6 d

17.1 q

24.3 q

173.8 s

77.9 t

120.6 d

177.3 s

36.0 t

36.8 t

212.7 s

42.8 t

43.7 d

35.9 t

69.1 d

45.6 d

35.8 d

35.0 s

21.3 t

36.7 t

37.2 t

212.7 s

42.2 t

43.6 d

26.6 t

21.5 t

41.7 d

36.7 d

35.4 s

21.2 t

41.5 t

49.8 s

85.8 s

42.2 t

73.2 d

58.1 d

16.9 q

22.7 q

168.8 s

75.7 t

120.0 d

174.5 s

37.6 t

38.9 t

214.5 s

43.7 t

45.1 d

37.2 t

70.3 d

46.8 d

33.8 d

36.0 s

30.7 t

75.9 d

57.2 s

87.0 s

44.4 t

73.6 d

55.0 d

10.5 q

22.7 q

173.1 s

77.7 t

32.4 t

27.5 t

100.4 s

34.5 t

40.1 d

37.1 t

70.9 d

46.4 d

35.0 d

35.1 s

21.4 t

42.0 t

49.4 s

86.6 s

42.4 t

73.7 d

58.5 d

17.0 q

23.2 q

169.0 s

75.6 t

119.7 d

174.7 s

47.7 q

(×2)

36.2 t

31.3 t

72.3 d

37.0 t

43.1 d

28.2 t

22.6 t *

43.0 d

37.4 d

35.9 s

21.9 t *

40.9 t

51.3 s

85.6 s

43.8 t

73.1 d

59.7 d

17.0 q

23.8 q

173.7 s

77.9 t

35.7 t

31.8 t

71.2 d

38.7 t

43.1 d

38.5 t

70.5 d

47.3 d

36.2 d

35.4 s

21.5 t

40.2 t

50.5 s

85.4 s

45.5 t

72.7 d

59.9 d

17.4 q

23.8 q

173.0 s

77.2 t

120.5 d

175.1 s

37.8 t

37.9 t

215.9 s

43.0 t

45.4 d

27.7 t

22.4 t

42.1 d

34.4 d

36.2 s

30.7 t

76.3 d

57.6 s

86.3 s

43.4 t

73.4 d

54.9 d

10.5 q

22.8 q

173.2 s

77.7 t

36.2 t

31.2 t

72.2 d

37.0 t

43.1 d

28.2 t

23.0 t

42.4 d

34.3 d

35.8 s

30.5 t

76.6 d

57.6 s

86.4 s

43.5 t

73.4 d

54.9 d

10.5 q

23.7 q

173.3 s

77.7 t

120.6 d

177.4 s

41.2 t

49.5 s

86.0 s

42.1 t

73.0 d

58.2 d

16.8 q

22.4 q

169.9 s

75.9 t

119.5 d

175.3 s

120.7 d

177.5 s

120.6 d

177.4 s

120.6 d

177.6 s

3-O-Me

-

* interchangeable.

Figure 3. Key HMBC and COSY correlations of the new metabolites (compounds

obtained from the microbial biotransformation of gitoxigenin by A. eureka 1E1BL1.

1, 3, 4, 6 and 8)

The molecular formula of

2 was determined as C₂₃H₃₂O₅based on the HR-ESI-MS

analysis (m/z 433.2227 [M + FA-H]⁻, calcd. 433.2231 for C₂₃H₃₂O₅) with eight indices of

Molecules 2021, 26, 3030

7 of 17

hydrogen deﬁciency (HD). Apart from the absence of hydroxymethine signal of C-3 and

the downﬁeld shift of carbon resonances belonging to A-ring, the characteristic signals

of the gitoxigenin were present for 2

. In the ¹³C-NMR spectrum, the resonance at δ_C

212.7 was evident for modiﬁcation of C-3 secondary alcohol to a ketone, substantiating the

increase in HD. Moreover, the HMBC correlations of C-3 with one of H₂-2 (δ_H2.32, dd,

J = 14.5; 6.4 Hz) and H₂-4 (δ_H2.62 m) resonances, justiﬁed the oxidation at C-3 (Figure 4).

Consequently, compound

2 was established as 3-oxo-gitoxigenin, a previously identiﬁed

compound [34,35].

Figure 4. Key HMBC and COSY correlations of the known metabolites (compounds

obtained from the microbial biotransformation of gitoxigenin by A. eureka 1E1BL1.

2, 5 and 7)

The HR-ESI-MS spectrum of

3 showed a major ion peak at m/z 419.2075 (calcd.

419.2075 for C₂₃H₃₂O₇[M-H]⁻) indicating a molecular formula of C₂₃H₃₂O₇. The disap-

pearance of the oxymethine signal of C-3 and the presence of a carbonyl carbon signal at

δ_C214.5 implied a similar modiﬁcation, as in

proved oxidation at C-3. In the spectrum, two additional low-ﬁeld resonances (δ_H3.91 and

δ_H3.36) were observed. Besides, two extra down-ﬁeld carbon signals at 70.3 and 75.9

were also noted. In order to deduce the oxygenation positions, the 2D NMR spectra were

studied thoroughly. Like , a monooxygenation at C-7 was conﬁrmed via inspection of the

1 and 2. Examination of the HMBC spectra

δ

1

COSY spectrum (H-7: δ_H3.91). The second hydroxy group was located at C-12 on the basis

of the HMBC spectrum, which showed a cross-peak between the C-12 (δ_C75.9) signal and

H-18 (δ_H0.89 s). The relative orientations of hydroxy groups at C-7 and C-12 were deter-

mined based on the 2D-NOESY data. The H-12 (δ_H3.36, dd J = 11.8, 4.1 Hz) signal showed

NOE correlation with

C-12 hydroxy as . Likewise, based on the NOE correlation observed between H-7 (δ_H3.91,

td J = 11.6; 11.2; 4.7 Hz) and -oriented H-9 (δ_H1.88, m), C-7 hydroxy group was deduced as

-oriented. Finally, the structure of was assigned as 7 ,12 -dihdyroxy-3-oxo-gitoxigenin.

The molecular formula of was found to be C₂₅H₃₈O₇based on the major ion peak at

m/z 449.2541 [M-H]⁻(calcd. 449.2545 for C₂₅H₃₈O₇[M-H]⁻). Inspection of the ¹H-NMR

spectrum of showed two O-methyl groups (δ_H3.12 and δ_H3.19) corresponding to the

α-oriented H-17 (δ_H3.58, d, J = 7.6 Hz) verifying the orientation of

β

α

β

3

β

4

same carbon resonance (δ_C47.7) in the HSQC spectrum suggesting a geminal dimethoxy

substitution. Based on the absence of a low-ﬁeld oxymethine signal due to H-3 in the

¹H-NMR spectrum, and the observation of a new quaternary signal (δ_C100.4) displaying

long-range correlations with δ_H3.12 and δ_H3.19 signals in the HMBC spectrum, a geminal

dimethoxy substitution at C-3 was deduced unambiguously. An additional low-ﬁeld

proton signal was also present at δ_H3.96 indicating an oxymethine group in the structure.

Examination of the COSY and HSQC spectra suggested the monooxygenation position

to be C-7, as in 3. This assumption was conﬁrmed by the HMBC experiment, which

showed the long-range correlations from H₂-6 (δ_H1.57 and 1.92) and H-8 (δ_H1.72) to

the 70.9 ppm resonance (C-7). The stereochemistry of the hydroxy group at C-7 was

established via 2D-NOESY spectrum. The observed cross peaks from H-7 (δ_H3.96, m) to

α

-oriented protons H-9 (δ_H1.54, m) and H-17 (δ_H2.91, dd, J = 7.0; 3.0 Hz) substantiated the

orientation of C-7 hydroxy as . Based on this evidence, the structure of was determined

as 3-dimethylacetal-7β-hydroxygitoxigenin.

β

4

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Compound

5

was identiﬁed as 3-epigitoxigenin by comparison of the spectroscopic

data with those previously reported by Kawaguchi et al. [36].

The major peak at m/z 405.2280 [M-H]⁻(calcd. 405.2283 for C₂₃H₃₄O₆[M-H]⁻)

in the HR-ESI-MS spectrum of compound

and

(3-epi-gitoxigenin), implying a hydroxylated analogue of either of them. The ¹H-

and ¹³C-NMR spectra of

were found to be very similar to those of , except for an

6 indicated 16 amu increase over gitoxigenin

5

6

5

additional proton at δ_H4.26 corresponding to a carbon signal at δ_C70.5 in the HSQC

spectrum (Tables 1 and 2). In the COSY spectrum, the spin system starting from the H-8

(δ_H1.95, m)/H-7 (δ_H4.26, m)/H₂-6 (δ_H1.80, m and 2.14 td, J = 12.2, 4.9 Hz), together with

the long-range correlations from C-7 (δ_C70.5) to H₂-6 and H-8 in the HMBC spectrum

(see Figure 3 ) helped us to determine the location of hydroxylation as C-7. The relative

conﬁguration of C-7 was clariﬁed via the interpretation of 2D-NOESY spectrum which

indicated a correlation between

appeared at δ_H2.67. In turn, the cross peak from

4.26, m) conﬁrmed the orientation of hydroxy group on C-7 as

peak from H-3 (δ_H3.85, m) to -oriented H-5 (δ_H1.51, m) in the 2D-NOESY spectrum was

α

-oriented H-16 (δ_H4.94, m) and one of H-15 protons

-oriented H-15 (δ_H2.67, m) to H-7 (δ_H

. Additionally, the cross

α

β

evident to verify the epimerization at C-3. Consequently, the structure of

6 was elucidated

as 7β-hydroxy-3-epi-gitoxigenin.

In the HR-ESI-MS of

H]⁻(calcd. 449.2181 for C₂₃H₃₂O₆[M + FA-H]⁻), indicating the same molecular weight

with that of . The presence of characteristic carbonyl carbon signal of C-3 ketone and

7, the major ion peak was observed at m/z 449.2179 [M + FA-

1

appearance of a downﬁeld proton resonance at δ_H3.40 (see Tables 1 and 2), corresponding

to δ_C76.3 signal in the HSQC spectrum, implied a monooxygenation in addition to ketone

formation on the cardenolide framework. In the HMBC spectrum, key cross peaks from

the carbon at δ_C76.3 to H₂-11 (δ_H1.30, m; 1.66, m) and H₃-18 (δ_H0.89, s) helped us to

determine the location of hydroxy group as C-12. The relative conﬁguration of C-12 was

established via 2D-NOESY data. The strong correlation between H-12 (δ_H3.40, dd, J = 11.8,

4.1 Hz) and

of C-12(OH) as

α

- oriented H-17 (δ_H3.59 d, J = 7.6 Hz), was evident to establish the orientation

. On the basis of these evidence, was identiﬁed as 3-oxo-diginatigenin, a

β

7

known compound [37].

The HR-ESI-MS spectrum of compound

8 provided a major ion peak at m/z 405.2280

[M-H]⁻(calcd. 405.2355 for C₂₃H₃₄O₆[M-H]⁻), indicating 16 amu increase over gitoxi-

genin and

resemblance with those of

trum of , in addition to the characteristic low-ﬁeld signals of

5

(3-epi-gitoxigenin). The ¹H- and ¹³C-NMR spectra of

rather than of gitoxigenin (Tables 1 and 2). The ¹H-NMR spec-

, an additional resonance at

8 showed substantial

5

8

5

δ_H3.34 was observed. In the ¹³C-NMR spectrum, the additional oxymethine signal at δ_C

76.6 undoubtedly demonstrated monohydroxylation on the skeleton, while a key HMBC

between the new oxymethine carbon signal and H₃-18 (δ_H0.87, s) helped us locating the

hydroxy group at C-12. In the NOESY spectrum, H-12 (δ_H3.34, m) showed correlation

with

. Also, the NOESY correlation between H-3 (δ_H3.59, m) and

veriﬁed the epimerization at C-3 indicating -orientation of C-3(OH). In conclusion, the

α

-oriented H-16 (δ_H4.60, td, J = 7.6, 1.7 Hz), establishing the orientation of OH-12 as

β

-oriented H-5 (δ_H1.45, m)

α

structure of 8 was elucidated as 3-epi-diginatigenin.

The microbial biotransformation study on gitoxigenin afforded ﬁve new cardenolide

derivatives

1, 3, 4, 6 and 8 together with 3 previously identiﬁed compounds 2, 5 and 7. The

structures and approximate yields were illustrated in Figure 5.

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Figure 5. Structures and yields of the metabolites obtained via microbial biotransformation of

gitoxigenin by A. eureka 1E1BL1.

Structurally, the 3-oxo metabolites of gitoxigenin

formation of 3-oxogitoxigenin by Gibberella fujikuroi and Fusarium sp. was demonstrated in

earlier biotransformation studies [34 36 38]. The structure was determined using paper

1–3 and 7 were mainly obtained. The

,

–

partition chromatography, IR and Kedde’s color reaction; however, NMR-based structural

characterization was absent. Also, absorption spectroscopy analysis of gitoxigenin and

3-oxo-gitoxigenin was reported with a series of related cardenolides. Likewise, other

known 3-keto derivative 3-oxodiginatigenin (

7), was fermentatively prepared from digi-

natigenin previously by Repke and Klesczewski; however, limited spectroscopic data was

available [37]. Thus, in this study,

NMR data for the ﬁrst time.

2 and 7 were characterized thoroughly based on the

Compounds

metabolites were earlier reported in independent biotransformation and semi-synthesis

studies [36 39]. Moreover, Repke and Samuels carried out an in vitro study with liver

5, 6 and 8 were 3-epi derivatives of gitoxigenin. Similar epimerization

–

tissue preparations from rats using digitoxigenin as substrate to examine the enzymatic

foundation of C-3 epimerization [40]. It was pointed out that the epimerization process of

cardiac steroids involved two stages: ﬁrst, oxidation of the substrate by diphosphopyridine

nucleotide (DPN)-dependent 3

3-keto intermediate by the triphosphopyridine nucleotide (TPN)-dependent 3

β

-hydroxy steroid dehydrogenases; second, reduction of the

-hydroxy

α

steroid dehydrogenases. Also, Hennebert et al. carried out docking experiments on

steroids with intention to explain the epimerization mechanism of dehydrogenases [41].

Considering the earlier reports and the formation of 3-keto metabolites chieﬂy in this

study, it is rational to state that the 3-epi derivatives are likely to be originated via 3-keto

intermediates by 3α-hydroxy dehydrogenases.

Finally, compound 4 exhibited an unusual structure with dimethyl acetal functionality

at C-3 reported here for the ﬁrst time. Previously, 3,3-dimethoxy analogs of two cardeno-

lides (uzarigenin and digitoxigenin) were prepared via semi-synthesis [42]. It is known

that aldehydes and ketones in acidic environment with the presence of methanol can form

dimethyl acetals. To make sure that 4 is not an artefact, LC-MS analyses were performed

for the samples of Day 6, 10, 14, 18 and the ﬁnal ethyl acetate (EtOAc) extract (Day 21)

used for isolation studies (see chromatograms S10 and S11 in the Supplementary Material).

In these experiments, the extracts were treated with neither acids nor methanol. Since

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compound

4

was detected in the last three time points (Day 14, 18 and 21), the formation of

dimethyl acetal was suggested to take place via biocatalysis. One plausible path for acetal

formation is P450 monoxygenase catalyzed geminal-diol formation at C-3. Accordingly,

Bell-Parikh and Guengerich demonstrated a slow oxidation reaction of ethanol to acetic

acid by human P450 2E1 and showed that gem-diol could be formed as an intermediate

within the reaction catalyzed via monooxygenation [43]. In another study, gem-diol for-

mation was catalyzed by CYP112, a bacterial P450 monooxygenase, as a part of microbial

gibberellin synthesis [44]. Based on the abovementioned research, a plausible stepwise

mechanism for acetal formation on the cardenolide core is put forward to be catalyzed by

P450 monooxygenases (Figure 6).

Figure 6. A plausible path for the formation of acetal group in 4.

Regarding hydroxylation, the monooxygenation took place only on the

β face of the

steroid nucleus involving C-7 ( and ) and C-12 ( and ) positions (Figure 6). In

1,

3,

4

6

7

8

other words, stereoselective monooxygenations were afforded by A. eureka endophyte on

the cardenolide skeleton. Most metabolites were monohydroxylated derivatives, whereas

the only dihydroxy product was compound 3.

Monooxygenations on the positions of C-7 and C-12 are frequently observed on

cardenolides [45]. Diginatigenin, also known as 12

ﬁrst by Murphy, as the aglycone of a cardiac glycoside diginatin isolated from Digitalis

lanata [46 47], and later Titus et al. isolated the compound as a biotransformation product

of gitoxigenin by Fusarium lini, reconﬁrming the structure [28]. Our biotransformation

β-hydroxygitoxigenin, was reported

,

studies on gitoxigenin by A. eureka yielded isolation of 3-epi analog of diginatigenin (8,

3-epi-diginatigenin), a previously unidentiﬁed compound.

Understanding the metabolism of drugs is vital for preclinical and clinical phases of

drug discovery processes being one of the most important factors affecting efﬁcacy/safety

rate. Cardenolides, for this purpose, were subjects of several microbial transformation stud-

ies, in which some oxidized metabolites were reported as tentative mammalian metabolites.

Repke et al. performed an investigation on animal metabolites of digitoxin. After intra-

venous administration of digitoxin into rats, urinary and fecal sampling for 48 h led to the

isolation of ten compounds, mainly exerting hydrolysis of the glycosidic bonds and 12

hydroxylations [37]. Another study was performed on humans by Ashley et al. through

digitoxin administration per os, and 12 -hydroxylated metabolites were determined in

β-

β

urine samples [48]. Our ﬁndings were consistent with both of these studies representing

12β-hydroxy metabolites.

2.3. MTT Cell Viability Assay

Cytotoxicity of the metabolites and substrate were evaluated in various cancer cell

lines (pancreatic: PANC-1, MIA PaCa-2; prostate: DU 145 and lung: A549) and two healthy

cell lines (lung: MRC-5 and kidney: HEK-293) by MTT cell viability assay (Table 3).

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Table 3. Cytotoxic activities of compounds 1–8 and the substrate gitoxigenin.

Compound

A549

DU 145

PANC-1

MIA PaCa-2

HEK-293

MRC-5

1

2

>10

3

4

>10

5

6

>10

7

8

>10

4.08

0.0210

0.1900

6.24

1.95

1.53

0.0387

>10

3.4

3.1

0.0350

0.5900

>10

7.6

0.0730

0.5870

>10

9.35

0.815

0.0173

0.8650

8.25

2.19

0.0426

0.6200

Gitoxigenin

Oleandrin

Doxorubicin

IC₅₀values are the concentration (µM) required 50% cell viability inhibition for a given compound with a 48 h treatment and were calculated

with using cell viability (%) formula via nonlinear regression analysis. Experiment was carried out in triplicate (n = 3). IC₅₀values were

given for A549 human lung cancer cell line, DU 145 human prostate cancer cell line, PANC-1 human pancreatic cancer cell line, Mia PaCa-2

pancreatic cancer cell line. HEK-293 human embryonic kidney cell line and MRC-5 human lung ﬁbroblast cell line as healthy cell lines.

Doxorubicin and oleandrin were used as positive controls.

Compound

an IC₅₀value of 6.24

against PANC-1, A549, and MIA PaCa-2 with 1.95, 8.25 and 3.4

These ﬁndings suggested that 12 -monohydroxylated derivatives

had higher cytotoxicity especially on PANC-1 cell line compared to the other metabolites.

Although, the IC₅₀values of for cancer cell lines were higher than the substrate and

the control groups (oleandrin and doxorubicin), slighter cytotoxic effect was observed for

healthy cell lines. Therefore, the new compound could be evaluated further because of

7

(3-oxodiginatigenin) exhibited cytotoxic activity against PANC-1 with

M, while 3-epidiginatigenin ( ) demonstrated higher bioactivity

M IC₅₀values, respectively.

and of gitoxigenin

µ

8

µ

β

7

8

its better therapeutic index and probably enhanced bioavailability.

Gitoxigenin was recently investigated for its cytotoxic activity against human can-

cer cell lines; TK-10 (renal adenocarcinoma), UACC-62 (melanoma) and MCF-7 (breast

adenocarcinoma), and the IC₅₀values were reported as 0.415

1.80 ± 0.32 µM, respectively [21]. The growth inhibitory effect of gitoxigenin on HeLa cells

was also reported as 2.25 g/mL [49], while in vitro inhibitory activity against human

nasopharynx carcinoma was presented with an ED₅₀value of 2.3 g/mL [50]. According

±

0.03, 2.83

±

0.17 and

µ

to our results, gitoxigenin exhibited cytotoxic activity against A549, DU 145, PANC-1, MIA

PaCa-2 cell lines with IC₅₀values ranging between 1.53 and 4.08 µM.

From the structure activity relationship perspective, as the C-3 epimers of gitoxigenin

have reduced cytotoxicity, one can state that the conﬁguration of C-3 (β-oriented OH

group), as in gitoxigenin, seems important for higher bioactivity. Furthermore, less toxic

biotransformation products obtained within this study point out to possible intermediates

of detoxiﬁcation mechanism for gitoxigenin.

Consequently, biotransformation attracts interest because of its advantages, viz. higher

speciﬁcity and efﬁciency. This study shows that A. eureka 1E1BL1 is a good choice for the

biotransformation of cardenolides. As incubation with a speciﬁc microorganism can

replace a series of reaction steps in chemical synthesis, ﬁndings presented here may offer a

biotechnological alternative to perform regio- and stereo-selective reactions. Furthermore,

the functional enzymes responsible for C-12β and C-7β stereoselective hydroxylations,

C-3 epimerization, C-3 oxidation or dimethyl acetal formation reactions on cardenolides,

might be isolated from A. eureka and then be used as biocatalysts in metabolite engineering

processes with the purpose of decreasing the cost of downstream processing.

3. Materials and Methods

3.1. General Experimental Procedures

Column chromatography studies were carried out using silica gel 60 (70-230 mesh,

Merck, Darmstadt, Germany) and LiChroprep^®RP-C18 (25–40

µm particle size, Merck)

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as adsorbents. A medium-pressure pump was used for vacuum liquid chromatography

(VLC) while RP-C₁₈column chromatography was accompanied by a peristaltic pump.

Analytical thin layer chromatography (TLC) was carried out on pre-coated silica gel 60 F₂₅₄

or RP-C₁₈F₂₅₄aluminium plates (20

and puriﬁcation steps have been checked by TLC plates on which spots were visualised by

spraying with 20% sulphuric acid in water and then heated at 105 C until visualized under

×

20 cm, layer thickness 0.2 mm, Merck). All isolation

◦

day light and examined under UV light (254 and 365 nm). Mass spectrometry analysis

were carried out on an Agilent 1200/6530 HR-ESI-MS instrument (Agilent Technologies,

Inc., Santa Clara, CA USA) in positive and negative modes. The LC-MS analysis was

performed on an Agilent 1260 Series HPLC system coupled with an Agilent G6550A

iFunnel Quadrupole Time-of-Flight with a Dual Spray Agilent Jet Stream electrospray

ionisation source (Agilent Technologies, Inc.). The instrument was operated in 2 GHz

Extended Dynamic Range mode with negative electrospray ionisation. The separation

was performed on a reversed phase Agilent Zorbax Extend column (4.6 mm

×

150 mm

×

3.5 m). The data was processed by Agilent MassHunter software B 06.00. 1D-, 2D-

µ

NMR spectra were recorded on a Varian AS 400 Mercury NMR spectrometer (Varian, Palo

Alto, CA, USA) and a Bruker DRX 500 (Bruker Biospin, Silberstreifen 4, Rheinstetten,

1

Germany). H-NMR spectra were recorded at 400/500 MHz, and the chemical shifts were

reported in units of parts per million (ppm) relative to the solvent peaks (CDCl₃:

CD₃OD: = 3.33 ppm or pyridine-d₅:

= 8.74; 7.58 and 7.22 ppm). ¹³C-NMR spectra were

recorded at 100 MHz, and the chemical shifts were reported in ppm relative to the solvent

peaks (CDCl₃: = 77.0, CD₃OD: 49.0 ppm or pyridine-d₅: 150.4; 135.9 and 123.9 ppm).

δ = 7.26,

δ

FT-IR (Fourier transform infrared) spectroscopic data were collected on a Perkin-Elmer

Spectrum 100 FT-IR Spectrometer (Perkin Elmer, Wellesley, MA, USA). Optical rotations

were measured in methanol using a AutoPol I polarimeter (Rudolph Research Analytical,

55 Newburgh Road Hackettstown, NJ, USA) at 18 ^◦C.

3.2. Plant Material

◦

0

00

Nerium oleander L. was harvested from Urla (Izmir, Turkey) in Nov 2019 (38 19 09.4 N

◦

0

00

˙

26 38 35.5 E). The plant material was identiﬁed by Prof. Dr. Erdal BEDIR (Department

of Bioengineering, IZTECH, Izmir, Turkey) and a voucher specimen is deposited at the

Herbarium of the Department of Pharmacognosy, Faculty of Pharmacy, Ege University,

Izmir, Turkey with the herbarium number of 1640.

3.3. Preparation of Gitoxigenin, the Substrate, by Acid-Catalysed Hydrolysis of Oleandrin

In the context of this study, oleandrin, which was recently isolated from dried leaves

of N. oleander according to a modiﬁed method of Ryer et al. [51] by our research team [33],

has been used as the main starting compound.

An acid-catalysed hydrolysis reaction of oleandrin was performed in order to obtain

gitoxigenin. Brieﬂy, 4 g of oleandrin was _◦dissolved in 1500 mL of 2 N triﬂuoroacetic

acid (TFA) in n-butanol and stirred at 200 C for 90 min under reﬂux. Hydrolysis was

carried out in four portions (1g oleandrin in 375 mL 2 N TFA in n-butanol per each).

The progression of the reaction was checked by TLC (Mobile system: 97:3 chloroform:

methanol) at every 30 min. At the end of 90 min, the reaction was ended neutralizing with

4 N KOH in water. Neutral reaction mixture was partitioned between chloroform and water

in order to remove salt and other inorganic impurities. Chloroform was evaporated at

◦

40 C and the organic residue (12.2 g) was loaded onto a silica gel column using a gradient

system of chloroform: isopropanol (100:0 to 95:5) to afford 80 fractions. Gitoxigenin with

slight impurities was obtained from the collected fractions of 51–72. Further puriﬁcation

was achieved by precipitation of Fr: 51–72 in acetonitrile (ACN) resulting in high purity

gitoxigenin (1.05 g, ca. 25% yield) as a white powder. Structural elucidation of gitoxigenin

was carried out by modern spectroscopic techniques (1D, 2D NMR and HR-ESI-MS).

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3.4. Microbial Biotransformation Procedure

Biotransformation of gitoxigenin was performed by Astragalus angustifolius-originated

endophytic fungus Alternaria eureka 1E1BL1, which was isolated and identiﬁed by our

group in 2016 [52]. The fungal strain stocks prepared as spore solutions in glycerol were

stored at

−

80 ^◦C, also potato dextrose agar (PDA) slants prepared and_◦stored at 4 ^◦C until

use. Fungi was grown on potato dextrose agar (PDA) Petri dishes at 25 C for 10 days, then

spore solution was prepared in 0.1% (v/v) Tween 80 and used to inoculate (2% v/v) ﬁve of

5 L Erlenmeyer ﬂasks containing 1.5 L of potato dextrose broth (PDB). After incubation

for 3 days, 1050 mg of gitoxigenin was added into the ﬂasks, and biotransformation was

carried out in a rotary shaker at 180 rpm, 25 ^◦C for 21 days. Biotransformation process

was followed by checking the ethyl acetate extracts of daily collected samples on TLC

plates [29].

3.5. Isolation and Puriﬁcation

After three weeks of biotransformation, fungal biomass was removed by ﬁltering

through Whatman No.1 ﬁlter paper on Buchner funnel under vacuum, and the broth was

extracted three times with equal volume of EtOAc. The organic phase was evaporated by

rotary vacuum evaporator at 40 ^◦C.

Initially, the crude EtOAc extract (8.7 g) was subjected to a silica gel column (175 g)

and eluted with a gradient system of CHCl₃: MeOH (98:2, 97:3, 95:5, 90:10). Following

collection of the main fractions (1–620), similar ones were combined together based on

their TLC proﬁles.

Firstly, the combined fractions of 15–16 (172 mg) were loaded onto an RP-C₁₈(17.5 g)

column which was eluted in gradient mode with H₂O: ACN (70:30 to 60:40) using a VLC

system. Subfractions 14–18 were pooled together and precipitated in cold ACN to obtain

1

(11.5 mg). The main fraction 14 (36.6 mg) was applied to a silica gel column (12 g) and

eluted with isocratic CHCl₃(100%) yielding 2 (4.6 mg).

Compound (2.6 mg) was isolated from the combined fractions of 528-572 (22.9 mg),

chromatographed over RP-C₁₈(17.5 g) by a VLC system using H₂O: ACN (80:20) for

elution. Compound (4.3 mg) was recovered from the combined fractions of 221–280

3

4

(71.3 mg) that was loaded onto a silica gel column (40 g) and eluted with a gradient system

of CHCl₃:MeOH (98:2, 97:3, 96:4, 90:10).

The combined fractions of 281–370 (40.4 mg) were applied to a silica gel column

(15.7 g) and eluted with an increasing gradient of n-hexane: EtOAc: MeOH (10:10:0.5 to

10:10:1.5). Subfractions 54–70 (14.6 mg) was subjected to a sequential fractionation using

silica gel (7 g) as stationary phase and CHCl₃: MeOH (97:3) as mobile phase to obtain

(2.3 mg).

5

Compounds 6 and 7 were isolated from the main fractions of 393–430 (37.4 mg), which

was chromatographed over an RP-C₁₈(17.5 g) VLC column and eluted with a gradient

system of H₂O:ACN (from 80:20 to 20:80). Subfraction 24 was further puriﬁed through

crystallization in cold ACN yielding

6

(1.9 mg), while

7 (2.7 mg) was obtained directly from

subfraction 22 in pure form. Compound

8

(1.6 mg) was isolated from the main fractions

of 604-619 (5.2 mg), which was fractionated on RP-C₁₈(17.5 g) by VLC system using a

gradient system of H₂O:ACN (from 90:10 to 80:20).

3.6. Compound Characterization

4-((3S,5R,10S,13R,14S,16S,17R)-3,14,16-Trihydroxy-10,13-dimethylhexadecahydro- 1H-cyclopenta

[a]phenanthren-17-yl) furan-2(5H)-one) (gitoxigenin). White powder. FT-IR (CHCl₃: MeOH):

3526, 3431, 2942, 2887, 1789, 1759, 1737, 1629, 1451, 1376, 1185, 1093, 1033, 888 cm⁻¹. HR-

ESI-M₋S (negative ion mode): m/z = 435.2387 [M + FA-H]⁻[Calculated for C₂₃H₃₄O₅[M +

◦

FA-H] 435.2388]. [α]

¹⁸= +14.7 (c 0.27, MeOH). ¹H-NMR (CD₃OD/drops of pyridine-d₅,

D

500 MHz): see Table 1; ¹³C-NMR (CD₃OD /drops of pyridine-d₅, 100 MHz): see Table 2.

Molecules 2021, 26, 3030

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(4-((5R,10S,13R,14S,16S,17R)-7,14,16-Trihydroxy-10,13-dimethyl-3-oxohexadecahydro-1H-

cyclopenta[a]phenanthren-17-yl)furan-2(5H)-one (7β-hydroxy-3-oxo-gitoxigenin, 1). White

needles. FT-IR (CHCl₃): 3405, 2942, 2873, 2156, 1739, 1712, 1631, 1617, 1449, 1341, 1284,

1110, 1051, 1032, 755 cm⁻¹. HR-ESI-MS (negative ion mode): m/z = 403.2123 [M

−

H]⁻

[Calculated for C₂₃H₃₂O₆[M-H]⁻403.2126]. [

α

]

¹⁸= +20.4^◦(c 0.25, MeOH). H-NMR

1

D

(CDCl₃/1 drop of CD₃OD, 400 MHz): see Table 1. ¹³C-NMR (CDCl₃/1 drop of CD₃OD,

100 MHz): see Table 2.

(4-((5R,10S,13R,14S,16S,17R)-14,16-Dihydroxy-10,13-dimethyl-3-oxohexadecahydro-1H-cyclopenta

[a]phenanthren-17-yl)furan-2(5H)-one (3-oxogitoxigenin,

IR (CHCl₃: MeOH): 3443, 2926, 2863, 1739, 1714, 1615, 1447, 1285, 1109, 1032, 895 cm⁻¹

HR-ESI-MS (negative ion mode): m/z = 433.2227 [M + FA-H]⁻[Calculated for C₂₃H₃₂O₅

2): White crystalline powder. FT-

.

[M + FA-H]⁻433.2231]. [ ¹⁸= +12.5^◦(c 0.4, MeOH). ¹H-NMR (CDCl₃, 400 MHz): see

α]

D

Table 1. ¹³C-NMR (CDCl₃, 100 MHz): see Table 2.

(4-((5R,10S,13S,14S,16S,17R)-7,12,14,16-Tetrahydroxy-10,13-dimethyl-3-oxohexadecahydro-1H-

cyclopenta[a]phenanthren-17-yl)furan-2(5H)-one (7β,12β-dihdyroxy-3-oxogitoxigenin, 3):

Yellowish-white oily substance. FT-IR (CHCl₃: MeOH): 3381, 2935, 2876, 1732, 1628,

1448, 1277, 1110, 1051, 1014 cm⁻¹. HR-ESI-MS (negative ion mode): m/z = 419.2075 [M

−

H]⁻[Calculated for C₂₃H₃₂O₇[M

−

H]⁻419.2075]. [ ¹⁸= +12.7^◦(c 0.24, MeOH).

α]

D

¹H-NMR (CD₃OD, 400 MHz): see Table 1. ¹³C-NMR (CD₃OD, 100 MHz): see Table 2.

(4-((5R,10S,13R,14S,16S,17R)-7,14,16-Trihydroxy-3,3-dimethoxy-10,13-dimethylhexadecahydro-1H-

cyclopenta[a]phenanthren-17-yl)furan-2(5H)-one (3-dimethylacetal-7β-hydroxygitoxigenin,

4

): White powder. FT-IR (CHCl₃): 3382, 2937, 2874, 1736, 1616, 1450, 1363, 1279, 1178, 1107,

1094, 1051, 1035, 877, 755 cm⁻¹. HR-ESI-MS (negative ion mode): m/z = 449.2541 [M +

FA-H]⁻[Calculated for C₂₅H₃₈O₇[M + FA-H]⁻449.2545]. [

α

]

= +20^◦(c 0.2, MeOH).

18

D

¹H- NMR (CDCl₃, 400 MHz): see Table 1. ¹³C-NMR (CDCl₃, 100 MHz): see Table 2.

(4-((3R,5R,10S,13R,14S,16S,17R)-3,14,16-Trihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]

phenanthren-17-yl)furan-2(5H)-one (3-epigitoxigenin,

MeOH): 3411, 2935, 2865, 2156, 1735, 1615, 1450, 1379, 1170, 1114, 1093, 1073, 1034 cm⁻¹

HR-ESI-MS (negative ion mode): m/z = 435.2385 [M + FA-H]⁻[Calculated for C₂₃H₃₄O₅

5): White needles. FT-IR (CHCl₃:

.

[M + FA-H]⁻435.2388]. [ ¹⁸= + 9.2^◦(c 0.11, MeOH). ¹H-NMR (CD₃OD, 400 MHz): see

α]

D

Table 1.; ¹³C-NMR (CD₃OD, 100 MHz): see Table 2.

(4-((3R,5S,10S,13R,14S,16S,17R)-3,7,14,16-Tetrahydroxy-10,13-dimethylhexadecahydro-1H-

cyclopenta[a]phenanthren-17-yl)furan-2(5H)-one (7β-hydroxy-3-epigitoxigenin, 6): White

needles. FT-IR (CHCl₃: MeOH): 3391, 2936, 2869, 2156, 1734, 1455, 1177, 1112, 1051,

774 cm⁻¹. HR-ESI-MS (negative ion mode): m/z = 405.2280 [M

−

H]⁻[Calculated for

18

1

C₂₃H₃₄O₆[M

−

H]⁻405.2283]. [

α

]

= +6.9^◦(c 0.29, MeOH). H-NMR (pyridine-d₅,

D

400 MHz): see Table 1. ¹³C-NMR (pyridine-d₅, 100 MHz): see Table 2.

(4-((5R,10S,13S,14S,16S,17R)-12,14,16-Trihydroxy-10,13-dimethyl-3-oxohexadecahydro-1H-

cyclopenta[a]phenanthren-17-yl)furan-2(5H)-one (3-oxodiginatigenin,

FT-IR (CHCl₃: MeOH): 3394, 2924, 2865, 1733, 1713, 1630, 1455, 1287, 1019, 775 cm⁻¹

HR-ESI-MS (negative ion mode): m/z = 449.2179 [M + FA-H]⁻[Calculated for C₂₃H₃₂O₆

7): White powder.

.

[M + FA-H]⁻449.2181]. [ ¹⁸= +21.9^◦(c 0.09, MeOH). ¹H-NMR (CD₃OD, 400 MHz): see

α]

D

Table 1. ¹³C- NMR (CD₃OD, 100 MHz): see Table 2.

(4-((3R,5R,10S,13S,14S,16S,17R)-3,12,14,16-Tetrahydroxy-10,13-dimethylhexadecahydro-1H-

cyclopenta[a]phenanthren-17-yl)furan-2(5H)-one (3-epidiginatigenin,

8): White powder. FT-IR

(CHCl₃: MeOH): 3390, 2929, 2863, 1732, 1614, 1559, 1451, 1074, 1035, 1027 cm⁻¹. HR-

ESI-MS (negative ion mode): m/z = 405.2280 [M

−

H]- [Calculated for C₂₃H₃₄O₆[M

−

18

H]⁻[405.2355]. [

α

]

= 0.0^◦(c 0.18, MeOH). ¹H-NMR (CD₃OD, 400 MHz): see Table 1.

D

¹³C-NMR (CD₃OD, 100 MHz): see Table 2.

Molecules 2021, 26, 3030

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3.7. MTT Assay

To see cytotoxic effects of the obtained metabolites, a standard MTT (3-(4,5-dimethylthiazol-

2-yl)-2,5-diphenyltetrazolium bromide) cell viability assay was performed on human cancer

cell lines PANC-1 (pancreatic, ATCC^®CRL-1469 ), MIA PaCa-2 (pancreatic, ATCC^®CRL-

™

1420

two healthy cell lines MRC-5 (lung, ATCC^®CCL-171

CRL-1573

) according to manufacturer’s instructions (M2128, Sigma-Aldrich^®). For this

™

), DU 145 (prostate, ATCC^®HTB-81

™

) and A549 (lung, ATCC^®CCL-185

) and HEK-293 (kidney, ATCC^®

™) and

™

purpose, cells at 80–90% conﬂuency were seeded to 96-well plates as 5-10x10³cells/well in

Eagle Minimal Essential Medium (EMEM) or Dulbecco Modiﬁed Eagle Medium (DMEM);

supplemented with 10% FBS and 1% L-glutamine depending on the cell type and incubated

in a humidiﬁed incubator at 37 ^◦C with 5% CO₂atmosphere for 24 h. After cell adhesion,

gitoxigenin and the metabolites were applied to the cells in triplicate with concentrations

ranging between 0.001 and 10 µM. After 48 h exposure to certain concentrations of the

compounds, medium was replaced with fresh one containing 10% MTT and plates were

incubated for 3 h in the dark for the conversion of tetrazolium salts to formazan crystals

by mitochondrial metabolism of viable cells. After incubation, formazan crystals were

dissolved in DMSO and absorbance of each well were measured at 570 nm via microplate

reader (Synergy

™ HTX- BioTek, Winooski, VT, USA). Doxorubicin (10 nM, 1 µM and 4 µM)

was used as positive control, whereas oleandrin (1 nM, 10 nM and 100 nM) was the second

positive control as a potent cardenolide.

All compounds were dissolved in DMSO (Sigma Aldrich, St. Louis, MO, USA) and

the ﬁnal DMSO concentration applied to cells was 0.02% (v/v), which is non-toxic to cells.

The IC₅₀values of the compounds were calculated by the normalization of optical densities

(OD) to negative control DMSO (0.02%, v/v).

Supplementary Materials: The following are available online. Supporting information for structural

elucidations (HR-ESI-MS; ¹H- and ¹³C-NMR and FT-IR spectroscopic data) of eight biotransformation

products (1–8) and substrate gitoxigenin; HPLC and LC-MS chromatograms of selected compounds

and crude EtOAc extract of biotransformation broth. Also, the ﬁgures of COSY, HSQC and HMBC

correlations are available for all compounds.

Author Contributions: Conceptualization and methodology, E.B.; investigation, Ç.K., G.D., G.K. and

M.K.; writing—original draft preparation, review and editing, Ç.K., G.D., G.K., M.K., H.Y. and E.B.;

supervision, E.B.; project administration, E.B.; resources, H.Y.; funding acquisition, Ç.K. All authors

have read and agreed to the published version of the manuscript.

Funding: This research was funded by Türkiye Bilimsel ve Teknolojik Aras¸tırma Kurumu (The

Scientiﬁc and Technological Research Council of Turkey, TUBITAK) [Grant number 119Z152].

Institutional Review Board Statement: Not applicable.

Informed Consent Statement: Not applicable.

Data Availability Statement: The data presented in this study are available in the text and Supple-

mentary Materials. Raw data will be provided by the corresponding author if requested.

Acknowledgments: This project is ﬁnancially supported by TUBITAK (project ID: 119Z152). We

are thankful to Salih GUNNAZ and Anzarulhaque Anwarulhaque for running NMR experiments,

˙

Ilhan Gürbüz for helping LC-MS studies, and Derya GÜLCEMAL for assisting optical rotation

measurements. We specially thank Eyüp Bilgi and Yig˘it Ege Çömlekçi for helping plant collection

and Özge Özs¸en for preliminary experiments.

Conﬂicts of Interest: The authors declare no conﬂict of interest.

Sample Availability: Samples of substrate gitoxigenin and compounds (1–8) are available from

the authors.

Molecules 2021, 26, 3030

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Article Doi

New cardenolides from biotransformation of gitoxigenin by the endophytic fungus alternaria eureka 1e1bl1: Characterization and cytotoxic activities

DOI: 10.3390/molecules26103030

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