preparative-cum-quantitative mass-directed analysis of swainsonine and its in situ ...

R E S EA RCH L E T T E R

Preparative-cum-quantitative mass-directed analysis ofswainsonine and its in situ activity against Sf-21 cell line

Digar Singh & Gurvinder Kaur

Department of Biotechnology, Indian Institute of Technology Guwahati, Guwahati, Assam, India

Correspondence: Gurvinder Kaur,

Department of Biotechnology, Indian Institute

of Technology Guwahati, Guwahati 781 039,

Assam, India. Tel.: +91 361 2582207;

fax: +91 361 2582249;

e-mail: [email protected]

Received 11 April 2013; revised 3 July 2013;

accepted 4 July 2013.

DOI: 10.1111/1574-6968.12214

Editor: Michael Bidochka

Keywords

swainsonine; mass-directed purification;

quantification; selected ion monitoring;

entomopathogen.

Abstract

Swainsonine is a polyhydroxy indolizidine alkaloid with various research and

potential therapeutic applications. In this work, swainsonine was partially

purified (2.5-folds) with acetone–methanol solvent system from Metarhizium

anisopliae fermentation broth. The partially purified broth was further

subjected to mass-directed preparative-cum-quantitative analysis. Swainsonine

was eluted as MS1 fraction [M + H]+ 174.36 � 0.21 at 4.91 � 0.04 min with

calculated yield of 7.85 � 1.59 lg mL�1 corresponding to 3.74 9 105 counts.

In situ antiproliferative activity of standard and purified swainsonine fractions

was tested against Spodoptera frugiperda, Sf-21 cell line with IC50 values of

2.96 lM and 3.28 lM, respectively, at 36 h. This analytical procedure for

purification and quantitative analysis of swainsonine may ensure its suitability

for routine laboratory studies and research.

Introduction

The entomopathogenic fungus Metarhizium anisopliae is a

well studied and applied species for microbial control of

insect pests (Schrank & Vainstein, 2010). Metarhizium spp.

produce a variety of secondary metabolites in several chem-

ical classes, including cytochalasins C and D, myroridins,

destruxins A, B and E, viridoxin, helvonic acid, 12-hydro-

xyovalicin, hydroxyfungerin, 7-desmethyl analogues of

fusarin C and (8Z)-fusarin C, serinocyclins A and B and

aurovertins. These metabolites are toxic to a broad range

of animals and microorganisms, including insects, fungi,

bacteria and viruses. One of the metabolites is swainsonine,

a trihydroxy indolizidine alkaloid with selective inhibition

property against Golgi a-mannosidase II that blocks the

abnormal formation of complex b-1, 6 branched N-linked

glycans, leading to reduced metastasis and tumour growth

(Thompson et al., 2012). Swainsonine is also described for

its apoptotic activities against cancerous A549 and C6

glioma cells, both in situ and ex situ (Li et al., 2012).

Swainsonine production and purification still remains a

challenge from both biological as well as expensive

synthetic routes. Earlier swainsonine was reportedly being

extracted and purified with various polar solvent systems,

physiological conditions and ion-exchange resins (Gardner

& Cook, 2010). However, the use of polar solvents such

as ethanol or water also imposes another problem of

co-extraction of broth components such as glucose, amino

acids and other such hydrophilic metabolites. Several

methods have been reported for the analysis and quantifi-

cation of swainsonine including capillary gas chromatogra-

phy, thin-layer chromatography, HPLC, LC-MS/MS and

HPLC with evaporative light-scattering detector (Yang

et al., 2012). Conventional HPLC methods have rarely been

applied to swainsonine analysis on account of reasons such

as (1) the lack of suitable chromophore groups rendering

its UV-visible detection almost impossible and (2) the

extreme hydrophilicity of swainsonine ensues the co-elution

of small polar molecules such as sugar or amino acids

(Molyneux et al., 2002). Moreover, preparative LC-MS

methods are advantageous over destructive low pH condi-

tions used in ion-exchange chromatography (Donaldson

et al., 1990). Hence, the development of sensitive, selective

and nondestructive method for the analysis of swainsonine

FEMS Microbiol Lett && (2013) 1–7 ª 2013 Federation of European Microbiological Societies.Published by John Wiley & Sons Ltd. All rights reserved

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has always been the prerequisite for its application studies.

Mass spectrometry is considered to be a specific and

universal detection method. Liquid chromatography–mass-

spectrometry-directed (LC-MSD) purification provides a

high-throughput rapid purification-cum-quantification of

diverse library compounds under selected ion monitoring

(SIM) mode (John et al., 2010; Chen et al., 2012).

Increasing sensitivity towards secondary metabolites

from fungal biological control agents has prompted

the toxicological risk assessment of metabolites pro-

duced by M. anisopliae (Skrobek & Butt, 2005). In situ

assays are important and useful tools in toxicity assess-

ment of various classes of environmental contaminants

including fungal metabolites because they significantly

reduce evaluation time and also provide information

about the mode of action of the toxicant (Fornelli

et al., 2004).

In the present study, we propose a novel mass-

directed purification-cum-quantification method for the

partially purified swainsonine and showed its entomo-

pathogenic activity against Spodoptera frugiperda (Sf-21)

cell line.

Materials and methods

Microorganism and Cultivation conditions

Metarhizium anisopliae ARSEF 1724 was procured from

ARS Collection of Entomopathogenic Fungal Cultures

(ARSEF) USDA, Ithaca, New York, and maintained in

Sabouraud dextrose agar slants at 4 °C. Basal oatmeal media

(60 mg mL�1) supplemented with glucose (20 mg mL�1) at

28 °C and 180 r.p.m. were used for the production of

swainsonine (Singh & Kaur, 2012).

Cell culture and treatments

Spodoptera frugiperda (Sf-21) cell line was procured from

National Centre for Cell Sciences (NCCS), Pune, India.

These were maintained as suspension (T-25 flask BD

biosciences) at nonhumidified, non-CO2 conditions and

at 28 °C in TNH-F media with 10% foetal bovine serum.

Cultures in the early stationary phase (typical cell density,

1.6 9 106 cells mL�1 with cell viability approximately

80%) were split every 3–4 days with a starting density of

4 9 105 cells mL�1.

Chemicals

The ingredients required for maintenance and swainsonine

production media were purchased from Hi-Media, India.

Swainsonine standard from M. anisopliae, a-D-mannosi-

dase from jack bean, p-nitrophenyl-a-D-mannopyranoside

and L-glutathione reduced were all obtained from Sigma-

Aldrich. All chemicals for purification were of HPLC grade

(Merck, Germany). All cell culture-related materials were

obtained from Sigma-Aldrich.

Swainsonine extraction and purification

The culture broth (85–90 mL) was centrifuged at

16 000 g for 10 min at 4 °C to separate the cells. The

supernatant was mixed with three different solvent

systems, viz. acetone (A), acetic acid (2%) in chloroform

(B) and ethanol (C) at the fixed proportion of 1 : 4. Cold

(�20 °C) and acidic (approximately pH 5.5) conditions

were employed up to 8 h for efficient extraction (Fellows

& Fleet, 1989). The mixture was immediately centrifuged

to remove the high molecular weight components. The

extract was then concentrated in vacuum rotary

evaporator (Ika HB 10) at 45 °C and freeze-dried before

final dissolution in 1 mL methanol.

Enzymatic quantification of swainsonine

The swainsonine titres in the culture supernatant, broth

extracts and purified samples were determined using

a-mannosidase inhibition assay (Sim & Perry, 1995).

Qualitative mass spectrometry–electro spray

ionization (MS-ESI+) analysis

Partially purified broth extracts were filtered (0.2 lm) and

MS-analysed (Agilent technologies Inc.). The MS-ESI+ ion

source parameters were adjusted for source and desolvation

gas temperatures at 100 and 250 °C, respectively. The

sampling, capillary and extraction cone voltages were set at

35 V, 3 KV and 3 V, respectively, with ion guide at 1 V.

The flow injection rate was fixed at 20 lL min�1. Instru-

ment control and data acquisition was performed with

Chemstation analytical software.

LC-MSD purification-cum-quantification

The MSD system (1260 Infinite series, Agilent technologies

Inc.) consists of binary solvent pump and autosampler with

thermostatted oven, reverse-phase C18 HPLC column

(21.2 9 250 mm 9 7 A°) and diode array detector

(DAD). Injection volume of 25 lL was loaded in all the

runs in LC column. Swainsonine was eluted using linear

gradient programme (100% water from 0 to 5 min and

100% acetonitrile from 5 to 20 min) at a flow rate of

10 mL min�1. The optimized capillary voltage 3 KV, frag-

mentor voltage 70 KV and collision energy 25 KV values

were applied. The mass spectrometer was run in SIM mode

for the mass range 173–174.36 � 0.21. Instrument control

FEMS Microbiol Lett && (2013) 1–7ª 2013 Federation of European Microbiological Societies.Published by John Wiley & Sons Ltd. All rights reserved

2 D. Singh & G. Kaur

and data acquisition was performed with Chemstation ana-

lytical software. LC chromatogram of total ion count (TIC)

vs. elution time (min) was analysed in commercial

MassHunter software (Agilent Technologies Inc.). The area

count for characteristic ion was extracted from the composite

total ion peak in SIM mode (Fattorusso & Taglialatela-Scafat-

i, 2007). Calibration curve of known concentration range,

1–8 lg mL�1, was constructed using standard swainsonine.

The observed [M + H]+ 174.36 � 0.21 intensities were

plotted against the corresponding concentrations.

Analytical LC-MS validation of swainsonine

MS1 fraction 174.36 � 0.21

Swainsonine mass fractions were further analysed by LC-

MS (Triple quad 6410, Agilent Technologies Inc.) with

analytical C18 column (4.6 9 250 mm 9 5 A°) fitted in

thermostatted oven and quaternary pump. The run

programme and solvent systems were all being the same

as above except the flow rate (1 mL min�1).

Evaluation of swainsonine cytotoxicity using

MTT assay

The MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltet-

razolium bromide) assay was used to describe the in situ

cytotoxicity of swainsonine against Sf-21 cell line (estab-

lished from ovaries of S. frugiperda). The standard and

MSD-purified swainsonine was tested at concentrations

ranging from 2.0 to 10.0 lM for 36 h with appropriate sol-

vent controls. A 100 lL of 1 9 105 cells mL�1 was seeded

into 96-well microtitre plates with 100 lL of swainsonine

of varying concentrations in complete growth media.

A 20 lL of MTT solution (5 mg mL�1 in PBS) was added

after the desired time points into each well, and the cells

were incubated for 4 h. The microtitre plate was centri-

fuged (250 g for 5 min), and the media were removed

gently before finally adding 100 lL of DMSO. The plate

was then rotated on an orbital shaker for 10 min to

dissolve the precipitate completely. The absorbance was

detected at 570 nm with reference wavelength of 660 nm

using microplate reader (Tecan, Infinite 200).

Statistical analysis

One-way ANOVA followed by Dunnett’s test of pairwise

multiple comparisons was used for MTT data analysis.

Cell viability plots were fitted in SPSS, version 11 (Science

Inc., Chicago, IL), with an exponential decay linear

combination equation to generate graphs. Each data point

represents mean of multiple wells, as mentioned in

legends to Fig. 5.

Microscopy

Microscopic analysis and imaging were performed at 409

magnification using inverted microscope (Nikon TS 100-F,

Japan).

Results and discussion

Swainsonine extraction and purification

Solvent Extraction, enzymatic quantification andMS analysis

A prior subtractive purification strategy was applied to

precipitate endogenous proteins, sugars and nucleotides,

etc. in fermentation broth using organic solvents under

chilled conditions. A maximum 2.5-fold purification was

achieved using solvent A (acetone), with 7.16 lg mL�1 of

swainsonine in extract supernatant. However, only 1.88-

and 1.16-fold purification of swainsonine was observed

with solvents B (5.19 lg mL�1) and C (3.35 lg mL�1),

respectively (Supporting Information, Table S1). The

purified fractions were further subjected to mass

spectrometry analysis and compared with mass spectrum

of swainsonine standard for characteristic MS1 counts

(Fig. 1a). The average swainsonine MS1 [M + H]+

174.36 � 0.21 ion counts were most abundant in solvent

A purified broth (Fig. 1b). The mass spectrum for solvent

B purified broth fraction was much noisy with number of

unknown higher mass fragments (Fig. 1c), whereas

solvent C purified fractions exhibited a comparatively

clear mass spectrum, but with insignificant average MS1

ion counts for swainsonine (Fig. 1d). There are certain

extra peaks, which may be hypothesized to be of glucose

units [M + Na]+, 180 + 23 = 203, or other such sodium

or formate adducts of higher sugars in fermentation

broth.

LC-MSD purification

The selective mass-directed purification can be used

directly with excellent recovery and purity without

involving the tedious packing of ion resins or prepacked

cartridges (Kagan et al., 2009; Gardner & Cook, 2010).

Here, the approach is based on unique fragmentation

patterns requiring either a precursor MS1 ion scan or a

neutral loss scan observed under MS/MS2 to generate the

species-specific quantification methodology as reported by

Chen et al., 2012;. Solvent A (acetone)-extracted broth

was selected for LC-MSD purification. Swainsonine MS1

fraction [M + H]+ 174.36 � 0.21 was eluted at an

average (te), 4.91 � 0.04 min, as the major peak.


Swainsonine purification and in situ applications 3

Fig. 2. DAD chromatogram of acetone-

extracted swainsonine fractions in LC-MSD

purification.

(a) (b)

(c) (d)

Fig. 1. Qualitative ESI-MS+ of (a) swainsonine standard, (b) acetone-extracted, (c) 2% acetic acid-chloroform-extracted and (d) ethanol-extracted

samples.

(a)

(b)

Fig. 3. LC-MS validation of MSD-purified

fractions (a) TIC chromatogram (b) ESI+ scan.



Swainsonine MS1 fractions were collected between

te = 4.41 and te = 4.78 min in vial 1 and te = 4.80 and

te = 5.15 min in vial 2. Native mass fractions of (m/z

173) were eluted between te = 5.66 and te = 6.14 min in

vial 4, but at negligible signal counts (Fig. 2). The puri-

fied fractions were further analysed and validated for

swainsonine purification with analytical LC-MS system.

Swainsonine was eluted as a single peak at

te = 6.38 � 0.04 min (Fig. 3a) with characteristic

m/z = 174.10 under MS1 scan showing 3.74 9 105 counts

(Fig. 3b).

Mass-directed LC-ESI-MS+ quantification

LC-MSD purification was simultaneously accompanied

with mass-directed quantification using the MS1 ion

counts [M + H]+ 174.36 � 0.21 in SIM mode. The stan-

dard curve with regression equation;

Y ¼ 1281314X � 1290157

where Y is MS1 ion counts (intensity) for swainsonine,

and X is swainsonine concentration (lg mL�1) with cor-

relation coefficient R of 0.99 (inset of Fig. 4). Swainso-

nine concentration was expressed as an average of three

replicate runs � SD, 7.85 � 1.59 lg mL�1 (Fig. 4). Thus,

the mass-directed technique was found to be more rapid

with high accuracy, selectivity and sensitivity for both

qualitative and quantitative analyses.

Fig. 4. Intensity ion counts vs. retention time

(min) curve for mass-directed quantification in

SIM mode. Calibration curve of swainsonine

for precursor ion [M + H]+ 174.36 � 0.21 as

an inset figure.

Fig. 5. MTT viability assay of Sf-21 cells measured after 36 h with

different doses of standard and purified swainsonine. One-way ANOVA

with Dunnett’s pairwise multiple comparison with control indicated

that purified swainsonine affected Sf-21 cell viability in a dose

dependent fashion (P < 0.001) with F = 405.64, signifying the test.

(a)

(b)

(c)Fig. 6. Phase contrast microscopic (409)

image of standard (2.96 lM) and purified

swainsonine (3.28 lM)-treated Sf-21 cells at

(a) 12 h, (b) 24 h and (c) 36 h.



Method validation for mass-directed LC-ESI-MS+quantification under SIM mode

Linearity

The selected ion count [M + H]+ 174.36 � 0.21 followed

linear relationship with the corresponding standard

concentration (1–8 lg mL�1) with correlation coefficient

(R) of 0.99. Thereafter, regression values altered

significantly from linearity (inset of Fig. 4). The limit of

detection (LOD) was defined as the lowest concentration

of swainsonine that gave an average signal-to-noise ratio

> 3 over three replicate injections based on MS1 ion

counts. The LOD for swainsonine was 0.25 lg mL�1.

Selectivity and precision

Selected mass fractions [M + H]+ 174.36 � 0.21 were

only observed in SIM mode. The relative standard

deviation (RSD) values for MS1 count intensity and

elution time were 0.79% and 4.04%, respectively, for

triplicate runs in LC-MSD purification and quantification

method (Fig. 4).

Accuracy

Swainsonine concentration derived from MSD

quantification was in agreement with that determined

from enzymatic assay method (Table. S1). The RSD value

between the two methods was found to be 6.5%.

In situ cytotoxicity of purified and standard swainsonine

against Sf-21 cell line

The MTT assay results showed that swainsonine

treatment caused inhibition of Sf-21 growth with as low

as 2 lM of standard and purified swainsonine for 36 h.

Further increase in swainsonine concentration (10 lM)

inhibited the growth by nearly 40% (Fig. 5). Thus, the

alkaloid treatment induced antiproliferative effects on

Sf-21 cells in a dose- and time-dependent manner for

36 h. Furthermore, cells viability data curve for both

standard and purified swainsonine was fitted to the

exponential decay linear combination equation at

R = 0.99 for the calculation of IC50:

Y ¼ Y0 þ ae�bx þ cX

where in case of purified swainsonine, Y is cell viability

(%), and X is swainsonine concentration (lM) with

coefficients Y0 = 54.14, a = 44.33, b = 1.07 and c = �1.51.

The observed IC50 values for purified and standard

swainsonine were nearly 3.28 lM and 2.96 lM, respec-

tively, at 36 h (Fig 5).

Phase contrast (409) images showed characteristic

membrane blebbing and released apoptotic bodies early

after 12 h (Fig. 6a), which became more distinct 24 h

onwards (Fig. 6b–c). The observed blebbing in cellular

membrane might affect the capacity of the cell to

transport and exchange substances with its environ-

ment. Generalized vacuolization and the toxic effect on

the mitochondria are the main symptoms of insect

intoxication by beauvericin (Valencia et al., 2011). To

date, there are no reports on the use of swainsonine

for insect cell viability assays, and hence, the present

study suggested its potential applications for pest

control.

Conclusions

A nondestructive purification-cum-quantification method

has been developed for swainsonine. Satisfactory validation

results were obtained in terms of precision and accuracy

using the LC-MSD. We also pointed out an in situ

target-specific biological activity of swainsonine against

S. frugiperda. The biological evaluation of swainsonine

produced by M. anisopliae provides better criteria to design

more effective formulations for pest management.

Acknowledgements

We thank Indian Institute of Technology Guwahati for

providing the experimental facilities and Council of

Scientific and Industrial research (CSIR), PUSA, New

Delhi, India, for providing the research fellowship. We

are also thankful to NCCS, Pune, India, for providing the

insect cell line. The authors have no conflict of interest.

Statement

The part of this work has been accepted in poster presen-

tation category at 5th congress of European microbiolo-

gists, FEMS 2013, Leipzig, Germany.

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Supporting Information

Additional Supporting Information may be found in the

online version of this article:

Table S1. Swainsonine extraction and purification with

organic solvents.



preparative-cum-quantitative mass-directed analysis of swainsonine and its in situ ...

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