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Chemical composition, and in vitroantibacterial and antifungal activityof an alkaloid extract from Crinumangustum Steud.Carmelina Iannelloa, Jaume Bastidab, Francesca Bonvicinic,Fabiana Antognonid, Giovanna Angela Gentilomic & Ferruccio Poliaa Department of Pharmacy and Biotechnology, University ofBologna, via Irnerio 42, 40137 Bologna, Italyb Department of Natural Products, Plant Biology and Soil Science,Faculty of Pharmacy, Av. Diagonal 643, 08028 Barcelona, Spainc Department of Pharmacy and Biotechnology, University ofBologna, Via Massarenti 9, 40138 Bologna, Italyd Department for Life Quality Studies, University of Bologna,Corso Augusto 237, 47921 Rimini, ItalyPublished online: 21 Jan 2014.

To cite this article: Carmelina Iannello, Jaume Bastida, Francesca Bonvicini, Fabiana Antognoni,Giovanna Angela Gentilomi & Ferruccio Poli (2014) Chemical composition, and in vitro antibacterialand antifungal activity of an alkaloid extract from Crinum angustum Steud., Natural ProductResearch: Formerly Natural Product Letters, 28:10, 704-710, DOI: 10.1080/14786419.2013.877903

To link to this article: http://dx.doi.org/10.1080/14786419.2013.877903

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Chemical composition, and in vitro antibacterial and antifungal activity ofan alkaloid extract from Crinum angustum Steud.

Carmelina Iannelloa, Jaume Bastidab, Francesca Bonvicinic, Fabiana Antognonid*,

Giovanna Angela Gentilomic and Ferruccio Polia

aDepartment of Pharmacy and Biotechnology, University of Bologna, via Irnerio 42, 40137 Bologna, Italy;bDepartment of Natural Products, Plant Biology and Soil Science, Faculty of Pharmacy, Av. Diagonal 643,08028 Barcelona, Spain; cDepartment of Pharmacy and Biotechnology, University of Bologna, ViaMassarenti 9, 40138 Bologna, Italy; dDepartment for Life Quality Studies, University of Bologna, CorsoAugusto 237, 47921 Rimini, Italy

(Received 15 October 2013; final version received 16 December 2013)

In the search for novel antibacterial agents against multidrug-resistant bacteria, analkaloid extract obtained from whole plants of Crinum angustum Steud., containing sixdifferent groups of alkaloids, was analysed by using gas chromatography/massspectrometry, and its in vitro activity against American Type Culture Collectionbacterial strains and clinical isolates was evaluated. The antimicrobial activity of theextract was tested against nine standard strains of microorganisms and two drug-resistant clinical isolates, methicillin-resistant Staphylococcus aureus and carbapene-mase-producing Klebsiella pneumoniae. The extract exhibited a significant activityagainst six of the examined strains of microorganisms. Antibacterial activity was morepronounced on Gram-positive bacteria than on Gram-negative bacteria, and IC50 valuesranged from 156 to 625mg/mL. Mycostatic activity against Candida albicans was alsofound, with an IC50 of 78mg/mL after 48 h of incubation . Promising results were alsoobtained from tests carried out on both clinical isolates investigated.

Keywords: Crinum angustum Steud; Amaryllidaceae; antimicrobial activity; alkaloids

1. Introduction

Plants of the Amaryllidaceae family are widely distributed in several countries, and include

about 860–1100 species in 85 genera growing largely in tropical and subtropical regions (Zhong

2007). These plants are well known not only for their ornamental value but also for the alkaloids

they produce. Some of these alkaloids are of particular interest for their wide range of biological

activities (Bastida et al. 2006). The genus Crinum, belonging to this family, includes

approximately 160 species present in warm temperate regions of the world (Mabberly 1990).

Cross-hybridisation of Crinum plants is rather widespread, mainly due to their particular interest

as ornamental plants. Indeed, several species of this genus are cultivated for both ornamental and

therapeutic purposes (Fennel & van Staden 2001).

In the search of new therapeutic strategies to cope with the development of multidrug

resistance (MDR) in pathogenic bacteria, which has recently created immense clinical problems

in the treatment of bacterial diseases (Boucher & Corey 2008), plant extracts may represent a

valuable source of compounds. In fact, several species of Crinum have already been proven for

their antibacterial and antifungal activities. For instance, the alkaloid crinamine isolated from

q 2014 Taylor & Francis

This manuscript was submitted at SIF 2013, National Congress of Societa Italiana di Fitochimica (ItalianPhytochemical Society), Gargnano (Italy).

*Corresponding author. Email: fabiana.antognoni@unibo.it

Natural Product Research, 2014

Vol. 28, No. 10, 704–710, http://dx.doi.org/10.1080/14786419.2013.877903

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the bulbs of Crinum jagus showed strong activity against Staphylococcus aureus (Adesanya

et al. 1992), while in vitro activity against Candida albicans was shown for Crinum macowanii

extracts (Gundidza 1986).

In this work, the chemical composition and antimicrobial activities of the alkaloid extract

obtained from whole plants of Crinum angustum, a hybrid between Crinum asiaticum var.

asiaticum and Crinum zeylanicum (Herbert 1837), were studied. The extract was tested against

reference bacterial strains and clinical isolates of methicillin-resistant S. aureus (MRSA) and

carbapenemase-producing Klebsiella pneumoniae (KPCs). Standard American Type Culture

Collection (ATCC) strains tested include S. aureus, Staphylococcus epidermidis, Streptococcus

pyogenes, Enterococcus faecalis, K. pneumoniae, Escherichia coli, Pseudomonas aeruginosa,

Proteus mirabilis and C. albicans.

2. Results and discussion

The analysis of the EtOAc extract by using gas chromatography (GC)/mass spectrometry (MS)

allowed the characterisation of six main types of alkaloids, namely the galanthindole-type,

lycorine-type, galanthamine-type, narciclasine-type, tazettine-type and haemanthamine-type.

Based on the percentage of TIC (total ion current) values, compounds present in the extract, in

decreasing order, were galanthindole 30.18% (1), lycorine 20.10% (2), N-demethylgalanthamine

11.73% (3), ismine 7.26% (4), N-formylnorgalanthamine 5.03% (5), 5,6-dihydrobicolorine

4.11% (6), galanthamine 4.03% (7), macronine 2.20% (8), crinamine 1.30% (9), trisphaeridine

1.24% (10) and vittatine 0.67% (11) (Figure 1).

As shown in Table 1, the alkaloid extract was found to be more active against Gram-positive

bacterial strains (S. aureus, S. epidermidis, S. pyogenes and E. faecalis) than against Gram-

negative bacterial strains (E. coli, K. pneumoniae, P. aeruginosa and P. mirabilis), with IC50

values ranging from 156 to 625mg/mL after 8 h incubation. These values were, in most cases,

lower than those found at 24 h incubation. Among Gram-negative bacterial strains, an inhibitory

activity was observed only on K. pneumoniae at 8 h incubation. The different activity against

Gram-positive and Gram-negative bacteria is not surprising, since major plant pathogens belong

to the latter group, and these species have evolved significant permeability barriers, which makes

the penetration of amphipathic compounds particularly difficult. Moreover, in Gram-negative

bacteria, a set of multidrug-resistance pumps efficiently extrudes amphipathic toxins across the

outer membrane (Lewis 2001). However, as a whole, the IC50 values which are reported here for

alkaloid extract are within the typical range of concentrations for plant compounds, which are

routinely classified as ‘antimicrobial’ on the basis of susceptibility tests that produce MICs in the

range of 100 to 1000mg/mL (Tegos et al. 2002). The difference in IC50 values towards the

different bacterial strains indicates that components of the alkaloid extract were selective in their

interaction with the pathogen, and this confirms the results reported by Cheesman et al. (2012).

A pronounced activity (IC50 78mg/mL) against C. albicans was found at 24 and 48 h incubation.

As regards the activity against clinical isolates (Table 2), the KPCs were inhibited by the extract

only at 8 h of incubation, while a bacteriostatic effect was found against the MRSA isolates with

IC50 values ranging from 156 to 1250mg/mL. At 8 h incubation, the IC50 values of the alkaloid

extract on MRSA clinical isolates were very similar to those found on ATCC strains (Tables 1

and 2).

A cytotoxicity test was carried out within the concentration range used for the antimicrobial

assays, and no cytotoxicity was observed (data not shown).

Based on the chemical structure of the molecules present in the extract and the biological

activities reported for these compounds, a hypothesis can be put forward regarding the major

candidates responsible for the antimicrobial activity. As regards lycorine, previous reports

demonstrated the lack of inhibitory effect of this alkaloid on the growth of S. aureus, E. coli and

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O

O CH2OH

NH3C

O

ON

OH

H

H

HO

1 2

NH

H3CO

OHO

O

OOH

HNH3C

3 4

N

H3CO

OHO

CHO

O

ONMe

5 6

MeO

O

NMe

OH

O

OO

O

OMe

NMe

H

7 8

O

ON

H H

H

OMe

OH

O

ON

9 10

O

ON

H H

H

OMe

H

11

Figure 1. Structure of compounds identified from the ethyl acetate extract of C. angustum Steud.(1) Galanthindole, (2) lycorine, (3) N-demethylgalanthamine, (4) ismine, (5) N-formylnorgalanthamine, (6)5,6-dihydrobicolorine, (7) galanthamine, (8) macronine, (9) crinamine, (10) trisphaeridine and (11) vittatine.

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P. aeruginosa, while showing an excellent antifungal activity on C. albicans (Evidente et al.

2004). Our results indicate that the Crinum alkaloid extract did not show any antibacterial

activity on E. coli and P. aeruginosa, showed a low activity on S. aureus (IC50 1.25mg/mL after

24 h incubation), while having a good antifungal activity on C. albicans. This can be explained

by the fact that both S. aureus and P. aeruginosa are able to transform lycorine into its inactive

metabolite 2-O-demethylungiminorine, instead of the active ungeremine (Ghosal et al. 1988).

Since a stimulatory effect on bacterial growth has been reported for 2-O-demethylungiminorine,

this can explain, in part, the high IC50 value found for the alkaloid extract on S. aureus after

24 h incubation.

The alkaloids of galanthamine-, narciclasine-, tazettine- and haemanthamine-type were also

present in Crinum alkaloid extract. As regards alkaloids belonging to the first three types, no

antibacterial or antifungal activities have hitherto been demonstrated, even though a wide array

of biological activities have been reported (Bastida et al. 2011). As regards alkaloids of the

haemanthamine-type, represented in this specific case by vittatine and crinamine, an

antibacterial activity was demonstrated on S. aureus and C. albicans (Evidente et al. 2004).

Vittatine was reported to be active against these strains (MIC values of 63 and 31mg/mL,

respectively), and an inhibitory activity against S. aureus was found for crinamine (Adesanya

et al. 1992). Thus, considering the results here obtained on Crinum alkaloid extract and data

from previous studies on purified alkaloids, it is likely that vittatine and crinamine did play a

Table 1. IC50 values of C. angustum Steud. on ATCC strains.

MicroorganismIC50 (8 h incubation)

(mg/mL)IC50 (24 h incubation)

(mg/mL)Positive control IC50 24 h

(mg/mL)

S. aureus 312 1250 1.0S. epidermidis 156 625 2.0S. pyogenes 312 312 4.0E. faecalis 625 – 4.0E. coli – – 4.0K. pneumoniae 625 – 4.0P. aeruginosa – – 8.0P. mirabilis – – 4.0C. albicansa No growth 78 (48 h: 78) 2.0

Note: –: no inhibition.a IC50 values for C. albicans ATCC 10231 were determined after 48 h incubation.

Table 2. IC50 values on the clinical isolates of C. angustum Steud.

MicroorganismIC50 (8 h incubation)

(mg/mL)IC50 (24 h incubation)

(mg/mL)

S. aureus, methicillin resistant (MRSA)a 625 625S. aureus, methicillin resistant (MRSA)b 625 1250S. aureus, methicillin resistant (MRSA)c 312 1250S. aureus, methicillin resistant (MRSA)d 312 625S. aureus, methicillin resistant (MRSA)e 78 156S. aureus, methicillin resistant (MRSA)f 312 625S. aureus, methicillin resistant (MRSA)g 156 625K. pneumoniae carbapenem resistant (KPC)1 312 –K. pneumoniae carbapenem resistant (KPC)2 625 –K. pneumoniae carbapenem resistant (KPC)3 – –

Notes: –: No inhibition; Letters and numbers in superscripts indicate clinical isolates from different patients.

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role in the antimicrobial activity of the extract. Besides these, galanthindole, a new type of

alkaloid with a non-fused indole ring (Unver et al. 2003), might also contribute to the inhibitory

activity. Indeed, the presence of an indole group within this molecule can support this hypothesis,

since an important role for this heterocyclic system in several biologically active molecules

exhibiting antimicrobial and antifungal capacity has been reported (Sharma et al. 2010).

It is worth noting that quite often the activity found for the total alkaloid extract is lower than

that of pure components, as a consequence of the fact that pure compounds are highly selective

in their interaction with the bacterial pathogens (Cheesman et al. 2012), thus being attenuated in

their potency when mixed with other components in a total extract.

3. Experimental

3.1. General procedure

The GC/MS analyses were performed in EI mode on a Hewlett Packard 6890 GC system.

A HP-5 MS column (30m £ 0.25mm £ 0.25mm) was used. The temperature programme was as

follows: 100–1808C at 158C min21, 1min hold at 1808C, 180–3008C at 58C min21 and 1min

hold at 3008C. Injector temperature was 2808C. The flow rate of the carrier gas (helium) was

0.8mL min21. A split ratio of 1:20 was used. Compounds were identified by comparing with

the NIST 05 database (NIST Mass Spectral Database, PC-Version 5.0, 2005), on the basis of

matching mass spectra and Kovats retention indexes (RI).

The following ATCC strains were used: S. aureus (ATCC 25923), S. epidermidis (ATCC

12228), E. faecalis (ATCC 29212), S. pyogenes (ATCC 19615), E. coli (ATCC 25922),

P. aeruginosa (ATCC 27853), K. pneumoniae (ATCC 9591), P. mirabilis (ATCC 9921) and

C. albicans (ATCC 10231).

The MDR Klebsiella pneumoniae clinical isolates (resistant to b-lactams, cephalosporins,

fluoroquinolones, aminoglycosides, macrolides, tetracyclines and carbapenems) and the MRSA

clinical isolates were from patients hospitalised in the St. Orsola-Malpighi University Hospital

in Bologna, Italy. Routine biochemical identification and antimicrobial susceptibility testing

were carried out using the Vitek2 semi-automated system (BioMerieux, Marcy l’Etoile, France)

3.2. Plant materials

C. angustum plants at the pre-flowering stage were collected in the greenhouse of the Botanical

Garden of Bologna University, Italy in January 2012. The plant was identified by Prof. Lucia

Conte, and a voucher specimen (No. BOLO0507744) has been deposited in the Herbarium of the

University Museum System (SMA), Bologna University, Italy.

3.3. Extraction and isolation

The crude methanolic extract from whole plants was purified using liquid–liquid separation.

Fresh bulbs and leaves (2.4 kg) were crushed together and exhaustively extracted with methanol

(MeOH) at room temperature for 72 h. The extract was evaporated under reduced pressure to

yield 114 g. This crude extract was acidified by dissolving it in 200mL of H2SO4 2% (v/v) and

neutral material was removed with diethyl ether (6 £ 200mL). The acidified solutions were then

basified with 25% NH4OH up to pH 10–11 and extracted with ethyl acetate (EtOAc, 8 £200mL) to give the final extract containing alkaloids (2.12 g). The extract was dried with

anhydrous Na2SO4, filtered and completely dried under reduced pressure. The extract yield, on a

fresh weight basis, represented approximately 0.09%.

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3.4. Antimicrobial assays

In vitro antimicrobial activity was determined by a broth micro-dilution method using 96-well

plates and according to National Committee for Clinical Laboratory Standards (NCCLS)-

recommended procedures. Mueller–Hinton (MH) broth (Bio-Rad) was used for all microbial

strains, except for S. pyogeneswhich was grown in MH broth supplemented with 3% lysed horse

blood (Bio-Rad).

Microbial suspensions at 5 £ 105 CFU/mL were incubated with twofold serial dilutions

starting from 2.5mg/mL of the extract sample (at least six dilutions). The corresponding controls

were included in all experiments: extract-free growth control, bacterial inoculum tested with

250mg/mL of gentamicin (Sigma-Aldrich) or fungal inoculum with 8mg/mL nystatin (Sigma-

Aldrich) (positive control), twofold serial dilutions of the tested extract inoculated with blank

medium without the inoculums, measuring both the background turbidity and the sterility of all

procedures (negative control), and twofold serial dilutions of DMSO (starting from 6.25% in

broth medium) inoculated with the microbial suspension assessing the interference effect of

the solvent (solvent control). The microplates were incubated at 378C for 8–24 h (and 48 h for

C. albicans) and microbial growth was estimated as optical density (OD at 630 nm) determined

by the Multiskan Ascent (Thermo Scientific) microplate reader. Results were evaluated as

follows: for each sample the net OD value was calculated (ODsample – ODnegativecontrol), then the

concentration required to produce 50% growth inhibition (IC50 value) was determined, relative

to the solvent control. The extract was assayed in quadruplicate and repeated on three different

days, for each microorganism.

4. Conclusions

In conclusion, this study has led to the identification of C. angustum Steud. as a possible source

of molecules with antimicrobial activity towards MDR bacteria. Studies are presently underway

to purify the extract in order to identify the components mainly responsible for the antibacterial

activity.

Acknowledgement

This research was supported by the Basic Research Program FARB of Ferruccio Poli (Finanziamenti diAteneo per la Ricerca di Base, 2013) from the University of Bologna. The authors declare no competingfinancial interest.

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