Pharmaceutical Biology

ISSN: 1388-0209 (Print) 1744-5116 (Online) Journal homepage: http://www.tandfonline.com/loi/iphb20

Bioactivity-guided isolation and structural

characterization of the antifungal compound,
plumbagin, from Nepenthes gracilis

Pei Shing Gwee, Kong Soo Khoo, Hean Chooi Ong & Nam Weng Sit

To cite this article: Pei Shing Gwee, Kong Soo Khoo, Hean Chooi Ong & Nam Weng Sit
(2014) Bioactivity-guided isolation and structural characterization of the antifungal compound,
plumbagin, from Nepenthes gracilis, Pharmaceutical Biology, 52:12, 1526-1531, DOI:
10.3109/13880209.2014.902083

To link to this article: http://dx.doi.org/10.3109/13880209.2014.902083

Published online: 15 Jul 2014.

Submit your article to this journal

Article views: 189

View related articles

View Crossmark data

Citing articles: 1 View citing articles

Full Terms & Conditions of access and use can be found at

http://www.tandfonline.com/action/journalInformation?journalCode=iphb20

Download by: [115.178.212.156] Date: 02 May 2017, At: 17:38

 http://informahealthcare.com/phb
ISSN 1388-0209 print/ISSN 1744-5116 online
Editor-in-Chief: John M. Pezzuto
Pharm Biol, 2014; 52(12): 15261531
! 2014 Informa UK Ltd. DOI: 10.3109/13880209.2014.902083

ORIGINAL ARTICLE

Bioactivity-guided isolation and structural characterization of the

antifungal compound, plumbagin, from Nepenthes gracilis
Pei Shing Gwee1, Kong Soo Khoo2, Hean Chooi Ong3, and Nam Weng Sit1
1
Department of Biomedical Science and 2Department of Chemical Science, Faculty of Science, Universiti Tunku Abdul Rahman, Bandar Barat,
Kampar, Perak, Malaysia, and 3Faculty of Science, Institute of Biological Sciences, University of Malaya, Kuala Lumpur, Malaysia

Abstract Keywords
Context: Despite several phytochemical studies of Nepenthes gracilis Korth (Nepenthaceae), Broth microdilution, extraction, fungicidal,
the biological activities of this pitcher plant remain to be explored. fungistatic, Nepenthaceae,
Objective: This study evaluates the antifungal activity of N. gracilis extracts, isolates, and naphthoquinone
characterizes its bioactive compound and evaluates the cytotoxicity of the isolated compound.
Materials and methods: Fresh leaves of N. gracilis were sequentially extracted. The fungistatic History
and fungicidal activities of the extracts were evaluated against six species of fungi of medical
importance using a colorimetric broth microdilution method. The most active extract was Received 12 September 2013
fractionated by liquidliquid partitioning and further purified by a preparative thin layer Accepted 2 March 2014
chromatography. Structural elucidation was carried out using FT-IR, GC-MS, and NMR. Published online 15 July 2014
Cytotoxicity testing against rhesus monkey kidney epithelial cells (LLC-MK2) was assessed by
a neutral red uptake (NRU) assay.
Results: The hexane extract, which showed the lowest minimum inhibitory concentration (MIC)
and minimum fungicidal concentration (MFC), both at 20 lg/mL against Candida albicans,
Issatchenkia orientalis, and Trichophyton mentagrophytes, was subjected to bioactivity-guided
fractionation. The isolated compound exhibited potent activity with the MIC values ranging
from 2 to 31 lg/mL against all the fungi. The active compound was identified as plumbagin
(5-hydroxy-2-methyl-naphthalene-1,4-dione). The 50% cytotoxicity concentration (CC50) of
plumbagin was 0.60 lg/mL.
Discussion and conclusion: The selectivity indices of plumbagin against all the fungi were less
than 1.0, indicating that plumbagin is more toxic to mammalian than fungal cells. This study
provides information on the antifungal properties of N. gracilis leaf extracts, as well as the
antifungal and cytotoxicity properties of plumbagin.

Introduction (Heitman et al., 2006). This poses great difficulties in

developing drugs that target fungi without affecting human
In the early 1900s, the pathogenic potential of certain
cells.
endemic fungi was first recognized when most of the
Nepenthes gracilis Korth. (Nepenthaceae) (Figure 1) is
systemic fungal diseases were described. A dramatic rise in
commonly known as monkey cup, which refers to the fact
invasive mycoses resulting from alterations in immune status
that monkeys occasionally drink rainwater from the pitchers
associated with the acquired immune deficiency syndrome
of these plants. It grows in nutrient-poor soils with a shallow
(AIDS) pandemic, organ and bone marrow transplantations,
root system. It has highly modified epiascidiate sword-shaped
and cancer chemotherapy (Kriengkauykiat et al., 2011;
leaves with tendrils that form the pitfall traps that efficiently
Pappas et al., 2010). These diseases cause morbidity and
capture, retain, and digest insect prey for nitrogen source
mortality in developed as well as developing countries as the
(Elison & Gotelli, 2001). Pitcher plants are used in traditional
availability of antifungal agents remains relatively limited and
medicine to treat dysentery, soothe inflamed skin, as an eye-
the emergence of widespread drug resistance (Denning &
wash, and as an astringent (Burkill, 1966). Extracts of
Hope, 2010; Miceli & Lee, 2011; Okeke et al., 2005).
Nepenthes mirabilis (Nepenthaceae) and Nepenthes ventri-
In addition, many antifungal drugs can have serious
cosa x maxima (Nepenthaceae) have been reported to possess
side effects due to the high degree of phylogenetic related-
antimicrobial activity against bacteria (Wiart et al., 2004)
ness between fungi and humans as both are eukaryotes
and plant pathogens (Shin et al., 2007a), respectively.
No studies have been reported on the antifungal activity of
Correspondence: Nam Weng Sit, Department of Biomedical Science, N. gracilis. This study evaluated the antifungal property of
Faculty of Science, Universiti Tunku Abdul Rahman, Bandar Barat,
31900 Kampar, Perak, Malaysia. Tel: +605 4688888x1016. Fax: +605 N. gracilis leaf extracts. The active compound was isolated
4661676. E-mail: sitnw@utar.edu.my and characterized by high performance thin layer
DOI: 10.3109/13880209.2014.902083
Figure 1. Nepenthes gracilis Korth.
chromatography (HPTLC), Fourier transform infrared spec-
troscopy (FT-IR), gas chromatography-mass spectrometry
(GC-MS), and nuclear magnetic resonance (NMR)
spectroscopy.
Materials and methods
Chemicals and reagents
The following chemicals and reagents used were of analytical
grade purity: hexane (Mallinckrodt Chemicals, Phillipsburg,
NJ), chloroform (System, Watsonville, CA), ethyl acetate
(R&M, Hampshire, UK), ethanol and glacial acetic acid
(PROCHEM, Alpharetta, GA), methanol (RCI Labscan,
Bangkok, Thailand), amphotericin B, Dulbeccos phosphate
buffered saline (DPBS), Dulbeccos modified Eagle medium
(DMEM), neutral red solution (3.3 g/L in DPBS), fetal bovine
serum (FBS), penicillinstreptomycin solution (100), tryp-
sin-EDTA solution (1), sodium bicarbonate, sodium chlor-
ide and p-iodonitrotetrazolium violet (Sigma-Aldrich, St.
Louis, MO), hexane and ethyl acetate of HPLC grade,
deuterium oxide (D2O), dimethyl sulphoxide (DMSO), gly-
cerol, potassium bromide (KBr), potato dextrose agar (PDA)
and sodium hydroxide pellets (Merck, Darmstadt, Germany),
RPMI-1640 medium supplemented with glutamine and
phenol red, without bicarbonate (MP Biomedicals,
Strasbourg, France), and 3-(N-morpholino) propanesulphonic
acid (MOPS) (Calbiochem, EMD Bioscience, La Jolla, CA).
Fungal strains tested
Candida albicans (ATCC 90028), Candida parapsilosis
(ATCC 22019), Issatchenkia orientalis (ATCC 6258),
Cryptococcus neoformans (ATCC 90112), Aspergillus
brasiliensis (ATCC 16404), and Trichophyton mentagro-
phytes (ATCC 9533) were purchased from the American
Type Culture Collection. The microorganisms were main-
tained on PDA at 4  C.
Antifungal activity of Nepenthes gracilis 1527
Cell culture
The rhesus monkey kidney epithelial cell line (LLC-MK2)
was a gift from Prof. Dr. Shamala Devi (Department of
Medical Microbiology, Faculty of Medicine, University of
Malaya, Malaysia). LLC-MK2 was propagated in DMEM
supplemented with 5% FBS at 37  C in a humidified
atmosphere with 5% CO2. For maintenance medium, the
FBS concentration was reduced to 1%.
Plant materials
Nepenthes gracilis specimens were purchased on 26
September 2010 from a nursery in Cameron Highlands,
Pahang, Malaysia. The identification of the species was
confirmed by one of the co-authors, Hean Chooi Ong, an
ethnobotanist from the University of Malaya. A voucher
specimen of N. gracilis (UTAR/FSC/10/016) was deposited at
the Faculty of Science, Universiti Tunku Abdul Rahman,
Perak Campus.
Preparation of extracts
Fresh leaves were washed thoroughly with tap water to
remove dirt and dust. The fresh leaves were weighed
(237.23 g) and cut into small pieces, blended, and sequentially
extracted using hexane, chloroform, ethyl acetate, ethanol,
methanol, and distilled water at room temperature with
agitation (120 rpm) using an orbital shaker (IKA-Werke KS
501, Staufen, Germany). Two cycles of extractions (1 d/cycle)
were performed for each solvent. The solvents were filtered
and evaporated using a rotary evaporator (Buchi Rota-vapor
R205, Brinkman, Switzerland) at 40  C. The water extracts
were lyophilized using a freeze-dryer (Martin Christ Alpha
1-4, LD Plus, Osterode, Germany). The samples were then re-
dissolved in a methanolwater mixture (2:1, v/v) at a
concentration of 10 mg/mL for the crude extracts, 1 mg/mL
for the isolated fractions, and 0.5 mg/mL for the isolated
compounds. All dissolved samples were filtered using a
0.45 lm syringe filters and stored at 20  C prior to analyses.
Preparation of fungal inocula
The preparation of broth medium and inoculum suspensions
was based on NCCLS/CLSI guidelines (NCCLS, 2002a,
2002b). The inoculum suspensions were prepared from fresh,
mature cultures grown on PDA. The suspensions were mixed
using a vortex mixer (VELP Scientifica, Milano, Italy) at
25 Hz for 15 s and adjusted to the optimal absorbance (A)
values (A 0.120.15 for Candida spp., I. orientalis and
C. neoformans; A 0.09  0.11 for A. brasiliensis, and
A 0.15  0.18 for T. mentagrophytes) at 530 nm. Further
dilutions using sterile saline solution (0.9% NaCl) were
performed to obtain the final working inoculum concentration
(15  103 CFU/mL for Candida spp. and I. orientalis;
15  104 CFU/mL for C. neoformans; 0.45  104 CFU/mL
for A. brasiliensis, and 1.26  104 CFU/mL for
T. mentagrophytes).
Screening for antifungal activity
The colorimetric broth microdilution method of Eloff (1998)
was modified and employed for screening the extracts for
1528 P. S. Gwee et al.
antifungal activity. This test utilized two-fold descending
concentrations of the extracts in 96-well microplates and a
reference antibiotic, with concentrations ranging from 2.5 to
0.02 mg/mL for the plant extracts, 0.25 to 0.002 mg/mL for
the fractions, 125 to 0.98 lg/mL for the isolated compounds,
and 8 to 0.063 lg/mL for amphotericin B. Growth, sterility,
and negative controls for the extracts and medium were
included. The 96-well microplates were incubated at 35  C for
48 h for Candida spp. and I. orientalis; 72 h for C. neoformans
and A. brasiliensis; and at 28  C for 7 d for T. mentagrophytes
(NCCLS, 2002a, 2002b). The colorimetric indicator, p-
iodonitrotetrazolium violet (0.4 mg/mL), was added and a
color change from colorless to red indicated a positive result.
The concentration at which the color remains clear was
recorded as the MIC value. The MFC was obtained by
inoculating 20 lL of the preparation that showed no evidence
of growth during the MIC determination assays on PDA.
The lowest concentration at which growth was not observed
was recorded as the MFC value. The tests were performed
in triplicate.
Isolation of the active compound from N. gracilis
The most active extract, the hexane extract, was subjected to
liquidliquid partitioning. The dry hexane extract (1.12 g) was
dissolved in 150 mL of hexane. This was partitioned with an
equal volume of water. The water layer was then extracted
using 150 mL of ethyl acetate. Each extraction was performed
three-times and the extracts were pooled together. The hexane
fraction and the ethyl acetate fraction were evaporated to
dryness by rotary evaporation, while the water fraction was
lyophilized. This yielded three fractions, i.e., hexane fraction
(F1), ethyl acetate fraction (F2), and water fraction (F3).
The dried samples for the F1, F2, and F3 fractions were re-
dissolved in a methanolwater mixture (2:1, v/v) for screening
for antifungal activity. The F1 fraction, which exhibited the
strongest antifungal activity, was further purified by prepara-
tive thin layer chromatography (PLC) using a hexaneethyl
acetate mixture (9:1, v/v) as the mobile phase. The F1 fraction
was re-dissolved in hexane (2 mg/mL) and applied (190 mL) as
a thin even layer horizontally above the solvent level on a
10  10 cm PLC plate. The development of PLC was
performed at room temperature. Two bands (assigned as B1
and B2) were removed from the packing material (silica gel 60
F254) of the PLC plate. The silica gels from those bands were
extracted twice with distilled water, followed by partitioning
with equal volume of hexane. The hexane solvent was
collected and evaporated using a rotary evaporator and
screened for antifungal activity. Following recovery of
components in B1, further purification was performed by re-
crystallization in hexane. This yielded yellow crystals, which
was identified as 5-hydroxy-2-methyl-naphthalene-1,4-dione
(plumbagin) based on spectroscopic data as well as by
comparison with the literature data.
Characterization of the active compound from
N. gracilis
Melting point was measured in a glass capillary tube by using
a melting point apparatus (Stuart SMP 10, Staffordshire, UK).
FT-IR spectroscopy was performed on KBr pellets containing
Pharm Biol, 2014; 52(12): 15261531
the sample (Perkin-Elmer Spectrum RX-1, Waltham, MA).
HPTLC analysis and PLC separation were performed on silica
gel plates (Kieselgel 60 F254, 0.2 mm; aluminum-backed and
1 mm; glass-backed, respectively, Merck, Germany) using a
semi-automatic sample applicator (Camag Linomat IV,
Switzerland). The HPTLC plate was scanned using a TLC
scanner (Camag TLC Scanner 3, Switzerland) and the data of
absorption signal spectra (200700 nm) were processed with
WinCATS software (Planar Chromatography Manager version
1.4.4, Camag, Switzerland) for purity assessment. The HPTLC
plates were photographed using a 12 bit charge-couple device
(CCD) digital camera (Camag TLC Visualizer, Switzerland)
under uniform illumination with white and UV light.
The mass spectrum of the isolated compound was obtained
by GC-MS (Shimadzu, QP2010 Plus, Tokyo, Japan) using
helium as the carrier gas. The working pressure was 99.8 kPa
and the flow rate was 1.46 mL/min. The GC-MS was fitted
with a 5% phenyl polysilphenylene-siloxane (BPX5) capillary
column of dimension 0.25 mm  30 m and the film thickness
was 0.25 lm. The injector port was maintained at 250  C
while the oven temperature gradient was 10  C/min from
80  C to 310  C with a total program time of 37 min. Electron
impact ionization method was used. The ion source and
interface temperatures were set at 200  C and 310  C,
respectively. The constituent was identified by comparing
with the mass spectral libraries (Wiley MS/NIST 05).
1
H-NMR (400 MHz) and 13C-NMR (100 MHz) spectra of
the isolated compound were recorded in deuterium oxide
(D2O) on a NMR instrument (JMN-ECX 400, JEOL, Tokyo,
Japan). All chemical shifts () were stated in parts per
million (ppm) with reference to tetramethylsilane (TMS) as
the internal standard and coupling constants (J) were
measured in Hz.
Determination of cytotoxic activity
LLC-MK2 cells (3  104 cells/well) were seeded in 96-well
microplates and incubated for 24 h at 37  C in a humidified
atmosphere with 5% CO2. After incubation, the cells were
treated with two-fold serially diluted concentrations of
plumbagin (0.056.25 lg/mL) for 72 h. The final concentra-
tion of the diluent (methanolwater mixture, 2:1, v/v) for
plumbagin was applied at the non-toxic concentration
(50.25%, v/v). Cell growth, sterility, and negative controls
were also included. The cell viability was evaluated using the
NRU assay according to the protocol of Repetto et al. (2008)
with modifications. The absorbance of the extracted neutral
red dye was measured at 540 nm using a microplate reader
(Tecan Infinite-M200, Mannedorf, Switzerland). The experi-
ment was performed in triplicate. The 50% cytotoxicity
concentration (CC50) of plumbagin on the cells was
determined from the plot of cell viability against concentra-
tions of plumbagin. The data were analyzed with one-way
ANOVA using Statistical Package for the Social Sciences
(SPSS) software (Version 15.0 for Windows, SPSS Inc.,
Chicago, IL) and the significance level was set at p50.05.
The toxicity of plumbagin to the LLC-MK2 cells and the
antifungal activity of plumbagin against each species of
fungus were compared by using the selectivity index (SI)
where SI CC50/MIC.
DOI: 10.3109/13880209.2014.902083 Antifungal activity of Nepenthes gracilis 1529
Table 1. MIC and MFC values of extracts from different solvents and isolated plumbagin from the leaves of N. gracilis against six types of fungi.

MIC (lg/mL) MFC (lg/mL)

Fungi Hex CH EA ET MT AQ B1 Ref Hex CH EA ET MT AQ B1
C. albicans 20 313625 625 313 313 2500 2 2 20 625 NA NA NA NA 2
C. parapsilosis 20 1250 625 313625 2500 NA 8 4 160 2500 NA NA NA 16
I. orientalis 20 313 313 160 80160 4080 2 4 20 313 NA NA NA NA 2
C. neoformans 20 313625 313 160 4080 40 4 0.125 160 1250 2500 2500 NA NA 8
A. brasiliensis 4080 1250 NA NA NA NA 31 2 160 NA 63
T. mentagrophytes 20 313 12502500 NA NA NA 2 8 20 313 2500 4

The results are shown as the mean or range values of triplicates. Hex, hexane extract; CH, chloroform extract; EA, ethyl acetate extract; ET, ethanol
extract; MT, methanol extract; AQ, water extract; B1, plumbagin; Ref, reference drug, amphotericin B; NA, no activity; , not tested since no MIC
value was obtained.

Table 2. MIC and MFC values of different fractions and compounds isolated from F1 of the hexane extract of
N. gracilis.

MIC (lg/mL) MFC (lg/mL)

Fraction/compound T. mentagrophytes C. neoformans T. mentagrophytes C. neoformans
Hexane fraction (F1) 8 16 8 16
B1 2 4 4 8
B2 62.5 125 125 NA
Ethyl acetate fraction (F2) 16 31 16 31
Water fraction (F3) NA NA
Ref 8 0.125

The results are shown as the mean values of triplicates. The meaning of the abbreviations are as follows: B1,
1st compound isolated from F1; B2, 2nd compound isolated from F1; Ref, reference drug, amphotericin B;
NA, no activity; -, not tested since no MIC value was obtained.

extract, F1 fraction exhibited the lowest MIC and MFC

Results
Following initial extraction, the highest percentage of yield
(w/w) was obtained from the ethanol extract (1.21%),
followed by chloroform, hexane, ethyl acetate, methanol,
and water extracts which gave 0.55, 0.47, 0.39, 0.21, and
0.05%, respectively. A total of 1.12 g of hexane extract was
obtained from 237.23 g of fresh leaves (0.47% yield). Further
fractionation of the hexane extract reduced to 0.783 g (0.33%
yield) of hexane fraction (F1), 0.247 g (0.10% yield) of ethyl
acetate fraction (F2), and 0.028 g (0.01% yield) of water
fraction (F3). Plumbagin (290 mg) was isolated from the
hexane fraction (0.12% yield).
The antifungal activities of extracts and plumbagin isolated
from N. gracilis against six types of fungi were evaluated
using a colorimetric broth microdilution method. The min-
imum inhibitory concentrations (MIC) and minimum fungi-
cidal concentrations (MFC) of various extracts and isolated
plumbagin are shown in Table 1. The MIC values ranged from
20 to 2500 lg/mL for the six extracts, from 2 to 31 lg/mL for
plumbagin, and from 0.125 to 8 lg/mL for amphotericin B
(positive control). The ethanol, methanol, and water extracts
did not exhibit any antifungal activity against the molds used
(A. brasiliensis and T. mentagrophytes), while the hexane
extract showed the lowest MIC value (20 lg/mL) against
C. albicans, C. parapsilosis, I. orientalis, C. neoformans, and
T. mentagrophytes. The hexane extract was, therefore,
selected for fractionation and isolation of active compounds.
Cryptococcus neoformans and T. mentagrophytes were
chosen as indicative microorganisms for the bioactivity-
guided fractionation. Following fractionation of hexane
values against the indicative microorganisms, ranging from
8 to 16 lg/mL, followed by F2 fraction at 1631 lg/mL while
F3 did not show any antifungal activity (Table 2). The F1
fraction contained the active compounds. Further isolation of
this fraction using preparative thin layer chromatography
(PLC) yielded two compounds (B1 and B2). The concentration
of B1 compound that inhibited the growth of the indicative
microorganisms was about 32-fold lower than that required
for compound B2 (Table 2). Compound B1 was thus subjected
to further characterization. Compound B1 effectively
inhibited the growth of all tested fungi and exerted a
fungicidal effect on all of them, with the MFC values ranging
from 2 to 63 lg/mL (Table 1). C. albicans and I. orientalis
were the most susceptible species with the lowest MFC value
of 2 lg/mL.
B1 compound was subjected to HPTLC, FT-IR, GC-MS,
and NMR analyses, and was subsequently identified as
plumbagin (5-hydroxy-2-methyl-naphthalene-1,4-dione).
Plumbagin forms yellow needle-shaped crystals and has a
melting point of 7879  C. It had an Rf of 0.34 on a HPTLC
silica gel 60 F245 plate developed with hexaneethyl acetate
(9:1, v/v) as the mobile phase. The UVVis absorption
spectrum displayed two absorption maxima at 260 nm and
425 nm, identical to those reported by Bothiraja et al. (2011).
The IR spectrum (KBr) showed the presence of OH
stretching at 3450 cm1, conjugated carbonyl groups at
1640 cm1, and the aromatic CH stretching at 803 cm1.
The GC-MS spectrum showed a molecular ion peak at
188.05 m/z and the spectrum obtained matched with the
reference standard plumbagin reported by Shin et al. (2007b).
1530 P. S. Gwee et al.
Figure 2. Cytotoxic effects of isolated
plumbagin from N. gracilis on rhesus
monkey kidney epithelial cells (LLC-MK2).
LLC-MK2 cells were treated with various
concentrations (0.056.25 lg/mL) of plum-
bagin for 72 h. The results represent
mean standard deviation of three replicates.
*Statistically significant (p50.05) with one-
way ANOVA.
The 1H- and 13C-NMR spectra confirmed the presence of
plumbagin with the molecular formula C11H8O3.
The viability of LLC-MK2 cells treated with various
concentrations of plumbagin as measured by NRU assay is
given in Figure 2. Plumbagin was found to be significantly
cytotoxic (p50.05) to the cells at concentrations ranging
from 0.78 to 6.25 lg/mL with the cell viability values less
than 10% after 72 h treatment. According to Figure 2,
the CC50 value of plumbagin on the LLC-MK2 cells was
0.60 mg/mL (3.19 lM). The cytotoxicity was not observed at
concentrations below 0.20 lg/mL as the percentages of cell
viability were approximately 100%. The SI values calculated
were 0.30 for C. albicans, I. orientalis, and T. mentagro-
phytes, 0.15 for C. neoformans, 0.08 for C. parapsilosis and
0.02 for A. brasiliensis.
Discussion
Several phenolic constituents, such as plumbagin, isoshina-
nolone, epishinanolone, shinanolone, quercetin, kaempferol,
and flavonoid gallate esters, have been isolated from the
leaves of N. gracilis (Aung et al., 2002; Fan et al., 2010).
However, reports of the pharmacological and biological
investigation of N. gracilis are scarce. To the best of our
knowledge, the present study is the first report to investigate
the antifungal property of N. gracilis leaf extracts against
fungi of medical important.
The six extracts obtained from the leaves of N. gracilis
exhibited antifungal activity against at least three types of
fungi (Table 1). The bioactivity-guided approach led to the
isolation of plumbagin from the hexane extract of N. gracilis.
Plumbagin is a secondary metabolite belonging to the
naphthoquinone group which is produced by many plants
belonging to the Caryophyllales order (Richer et al., 2002)
such as Droseraceae (Budzianowski, 2000), Plumbaginaceae
(Bothiraja et al., 2011), Ebenaceae (Evans et al., 1999), and
Nepenthaceae (Aung et al., 2002). A wide range of biological
properties of plumbagin such as anticancer (Kawiak et al.,
2007), antiviral (Perez-Sacau et al., 2003), anti-inflamma-
tory, antiplatelet, antiallergic (Lien et al., 1996), antimalarial
(Biot et al., 2004), and antibacterial (Cai et al., 2000) have
been reported. Dzoyem et al. (2007) reported the antifungal
Pharm Biol, 2014; 52(12): 15261531
activity of plumbagin isolated from the stem bark of
Diospyros crassiflora. In this study, the concentration of
plumbagin that inhibited the growth of I. orientalis, and
T. mentagrophytes was about 2-fold and 4-fold lower,
respectively, than that required for amphotericin B (Table 1).
The values of the selectivity indices of the isolated
plumbagin against all the fungi were less than 1.0, indicating
plumbagin is more toxic to mammalian than fungal cells.
Seshadri et al. (2011) have shown that the cytotoxic effect of
plumbagin in human peripheral blood lymphocytes was at
least two-fold higher than that of juglone (5-hydroxy-1,4-
naphthalenedione). Plumbagin at 5 mM was proven to be non-
cytotoxic to the resting mouse lymphocytes, but at the
concentration of 2 mM was found to induce apoptosis in
human resting lymphocytes, indicating the species-specific
differences in the activity of plumbagin (Checker et al.,
2009). Two major mechanisms have been proposed for
plumbagin cytotoxic action in various biological systems.
First mechanism is associated with the excessive generation
of reactive oxygen species (ROS) such as superoxide radicals,
singlet oxygen, and hydrogen peroxide (Castro et al., 2008;
Seung et al., 1998). Second mechanism involved the redox
and oxidation cycle of quinones namely redox cycles
which lead to their ability to act as potent electrophiles to
inhibit the electron transportation, as uncouplers of oxidative
phosphorylation, as intercalating agent in the DNA double
helix and as bio-reductive alkylating agents in the biomol-
ecules (Babula et al., 2009). The redox cycling and free
radical forming was identified as the main mode of actions
for plumbagin.
In animal studies, it has been noted that treatment with
plumbagin causes reproductive toxicity (Bhargava, 1984;
Premakumari et al., 1977). The oral bioavailability of
plumbagin in rat was less than 40%, due to its high
lipophilicity and insolubility in water (Hsieh et al., 2006).
Consequently, a large and frequent dose is necessary to
achieve the optimum therapeutic efficacy and this may lead to
severe side effects. The toxicity of plumbagin is a major
obstacle to its clinical application. The data reported herein
are important, taking into account the medical importance of
the studied fungi and that this plant species is traditionally
consumed or applied externally, without considering the
DOI: 10.3109/13880209.2014.902083
presence of toxic components such as plumbagin. Thus, the
pharmacological study of medicinal plants remains important
to provide evidence with scientific basis for the continued
traditional application of plants and understanding of a plants
efficacy as well as toxicity.
Conclusions
Plumbagin plays a significant role in the antifungal activity of
N. gracilis. The present study demonstrated that the leaves of
this plant could serve as an antifungal agent but caution
should be taken as plumbagin is a potentially cytotoxic
compound.
Acknowledgements
The authors thank Universiti Tunku Abdul Rahman for a
research grant (UTARRF Vote No. 6200/S07) which sup-
ported the M.Sc. candidature of Pei Shing Gwee.
Declaration of interest
The authors declare no conflict of interest.
References
Aung HH, Chia LS, Goh NK, et al. (2002). Phenolic constituents from
the leaves of the carnivorous plant Nepenthes gracilis. Fitoterapia 73:
4457.
Babula P, Adam V, Havel L, Kizek R. (2009). Noteworthy secondary
metabolites napthoquinones their occurrence, pharmacological
properties and analysis. Curr Pharm Anal 5:4768.
Bhargava SK. (1984). Effects of plumbagin on reproductive function of
male dog. Ind J Exp Biol 22:1536.
Biot C, Bauer H, Schirmer R, Davioud-Charvet E. (2004). 5-Substituted
tetrazoles as bioisosteres of carboxylic acids. Bioisosterism and
mechanistic studies on glutathione reductase inhibitors as antimalar-
ials. J Med Chem 47:597283.
Bothiraja C, Joshi PP, Dama GY, Pawar AP. (2011). Rapid method for
isolation of plumbagin, an alternative medicine from roots of
Plumbago zeylanica. Eur J Integrative Med 3:3942.
Budzianowski J. (2000). Napthoquinone glucosides of Drosera gigantea
from in vitro cultures. Planta Med 66:6679.
Burkill IH. (1966). A Dictionary of the Economic Products of Malay
Peninsula, Vol. 2. Kuala Lumpur: Ministry of Agriculture and
Cooperatives.
Cai L, Wei G-X, Van der Bijl P, Wu CD. (2000). Namibian chewing
stick, Diospyros lycioides, contain antibacterial compounds against
oral pathogens. J Agric Food Chem 48:90914.
Castro FAV, Mariani D, Panek AD, et al. (2008). Cytotoxicity
mechanism of two naphthoquinones (menadione and plumbagin) in
Saccharomyces cerevisiae. PLoS One [Online]. Available from: http://
www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.
0003999#pone-0003999-g003 [last accessed 5 Jan 2014].
Checker R, Sharma D, Sandur SK, et al. (2009). Anti-inflammatory
effects of plumbagin are mediated by inhibition of NF-kappaB
activation in lymphocytes. Int Immunopharmacol 9:94958.
Denning DW, Hope WW. (2010). Therapy for fungal diseases:
Opportunities and priorities. Trends Microbiol 18:195204.
Dzoyem JP, Tangmouo JG, Lontsi D, et al. (2007). In vitro antifungal
activity of extract and plumbagin from the stem bark of Diospyros
crassiflora Hiern (Ebenaceae). Phytother Res 21:6714.
Elison AM, Gotelli NJ. (2001). Evolutionary ecology of carnivorous
plants. Trends Ecol Evol 16:6239.
Antifungal activity of Nepenthes gracilis 1531
Eloff JN. (1998). A sensitive and quick microplate method to determine
the minimal inhibitory concentration of plant extracts for bacteria.
Planta Med 64:71114.
Evans PH, Bowers WS, Litaudon M, Sevenet T. (1999). Plumbagin from
Diospyros olen. Molecules 4:M93.
Fan DH, Wang H, Zhi D, Shen YM. (2010). CE analysis of endogenous
flavonoid gallate esters from Nepenthes gracilis (Nepenthaceae).
Chromatographia 72:101316.
Heitman J, Filler SG, Edwards Jr JE, Mitchell AP. (2006). Molecular
Principles of Fungal Pathogenesis. Washington, USA: ASM Press,
311.
Hsieh YJ, Lin LC, Tsai TH. (2006). Measurement and pharmacokinetic
study of plumbagin in a conscious freely moving rat using liquid
chromatography/tandem mass spectrometry. J Chromatogr B Analyt
Technol Biomed Life Sci 844:15.
Kawiak A, Piosik J, Stasilojc G, et al. (2007). Induction of apoptosis by
plumbagin through reactive oxygen species-mediated inhibition of
topoisomerase II. Toxicol Appl Pharmacol 223:26776.
Kriengkauykiat J, Ito JI, Dadwal SS. (2011). Epidemiology and
treatment approaches in management of invasive fungal infections.
Clin Epidemiol 3:17591.
Lien J, Huang L, Wang J, et al. (1996). Synthesis and antiplatelet, anti-
inflammatory and antiallergic activities of 2,3-disubstituted 1,4-
napthoquinones. Chem Pharm Bull 44:11817.
Miceli MH, Lee SA. (2011). Emerging Moulds: Epidemiological Trends
and Antifungal Resistance. Mycoses [Online]. Available from: http://
onlinelibrary.wiley.com/doi/10.1111/j.1439-0507.2011.02032.x/pdf
[last accessed 9 Feb 2012].
National Committee on Clinical Laboratory Standards (2002a).
Reference Method for Broth Dilution Antifungal Susceptibility
Testing of Yeasts; Approved Standard, NCCLS Document M27-A2,
2nd ed. Pennsylvania: NCCLS.
National Committee on Clinical Laboratory Standards (2002b).
Reference Method for Broth Dilution Antifungal Susceptibility
Testing of Filamentous Fungi; Approved Standard, NCCLS
Document M38-A. Pennsylvania: NCCLS.
Okeke IN, Laxmaninarayan R, Bhutta ZA, et al. (2005). Antimicrobial
resistance in developing countries. Part 1. Recent trends and current
status. Lancet Infect Dis 12:48193.
Pappas PG, Alexander BD, Andes DR, et al. (2010). Invasive fungal
infections among organ transplant recipients: Results of the
Transplant-Associated Infection Surveillance Network
(TRANSNET). Clin Infect Dis 50:110111.
Perez-Sacau E, Estevez-Braun A, Ravelo A, et al. (2003). Inhibitory
effects of lapachol derivatives on Epstein-barr virus activation. Bioorg
Med Chem 11:4838.
Premakumari P, Rathinam K, Santhakumari G. (1977). Antifertility
activity of plumbagin. Ind J Med Res 65:82938.
Repetto G, del Peso A, Zurita JL. (2008). Neutral red uptake assay for the
estimation of cell viability/cytotoxicity. Nat Protoc 3:112531.
Richer H, Hamm A, Bringmann G. (2002). Nepenthes insignis uses a C-
2-portion of the carbon skeleton of L-alanine acquired via its
carnivores organs, to build up the allelochemical plumbagin.
Phytochemistry 59:6039.
Seshadri P, Rajaram A, Rajaram R. (2011). Plumbagin and juglone
induce caspase-3-dependent apoptosis involving the mitochondria
through ROS generation in human peripheral blood lymphocytes. Free
Radic Biol Med 51:2090107.
Seung S, Lee JY, Lee MY, et al. (1998). The relative importance of
oxidative stress versus arylation in the mechanism of quinone-induced
cytotoxicity to platelets. Chem Biol Interact 113:13344.
Shin KS, Lee S, Cha BJ. (2007a). Suppression of phytopathogenic fungi
by hexane extract of Nepenthes ventricosa x maxima leaf. Fitoterapia
78:5856.
Shin KS, Lee S, Cha BJ. (2007b). Antifungal activity of plumbagin
purified from leaves of Nepenthes ventricosa x maxima against
phytopathogenic fungi. Plant Pathol J 23:11315.
Wiart C, Mogana S, Khalifah S, et al. (2004). Antimicrobial screening of
plants used for traditional medicine in the state of Perak, Peninsular
Malaysia. Fitoterapia 75:6873.