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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
ORIGINAL ARTICLE
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.
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
Figure 1. Nepenthes gracilis Korth. 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
chromatography (HPTLC), Fourier transform infrared spec-
extracted using hexane, chloroform, ethyl acetate, ethanol,
troscopy (FT-IR), gas chromatography-mass spectrometry
methanol, and distilled water at room temperature with
(GC-MS), and nuclear magnetic resonance (NMR)
agitation (120 rpm) using an orbital shaker (IKA-Werke KS
spectroscopy. 501, Staufen, Germany). Two cycles of extractions (1 d/cycle)
were performed for each solvent. The solvents were filtered
Materials and methods
and evaporated using a rotary evaporator (Buchi Rota-vapor
Chemicals and reagents R205, Brinkman, Switzerland) at 40 C. The water extracts
were lyophilized using a freeze-dryer (Martin Christ Alpha
The following chemicals and reagents used were of analytical
1-4, LD Plus, Osterode, Germany). The samples were then re-
grade purity: hexane (Mallinckrodt Chemicals, Phillipsburg,
dissolved in a methanolwater mixture (2:1, v/v) at a
NJ), chloroform (System, Watsonville, CA), ethyl acetate
(R&M, Hampshire, UK), ethanol and glacial acetic acid concentration of 10 mg/mL for the crude extracts, 1 mg/mL
for the isolated fractions, and 0.5 mg/mL for the isolated
(PROCHEM, Alpharetta, GA), methanol (RCI Labscan,
compounds. All dissolved samples were filtered using a
Bangkok, Thailand), amphotericin B, Dulbeccos phosphate
0.45 lm syringe filters and stored at 20 C prior to analyses.
buffered saline (DPBS), Dulbeccos modified Eagle medium
(DMEM), neutral red solution (3.3 g/L in DPBS), fetal bovine
Preparation of fungal inocula
serum (FBS), penicillinstreptomycin solution (100), tryp-
sin-EDTA solution (1), sodium bicarbonate, sodium chlor- The preparation of broth medium and inoculum suspensions
ide and p-iodonitrotetrazolium violet (Sigma-Aldrich, St. was based on NCCLS/CLSI guidelines (NCCLS, 2002a,
Louis, MO), hexane and ethyl acetate of HPLC grade, 2002b). The inoculum suspensions were prepared from fresh,
deuterium oxide (D2O), dimethyl sulphoxide (DMSO), gly- mature cultures grown on PDA. The suspensions were mixed
cerol, potassium bromide (KBr), potato dextrose agar (PDA) using a vortex mixer (VELP Scientifica, Milano, Italy) at
and sodium hydroxide pellets (Merck, Darmstadt, Germany), 25 Hz for 15 s and adjusted to the optimal absorbance (A)
RPMI-1640 medium supplemented with glutamine and values (A 0.120.15 for Candida spp., I. orientalis and
phenol red, without bicarbonate (MP Biomedicals, C. neoformans; A 0.09 0.11 for A. brasiliensis, and
Strasbourg, France), and 3-(N-morpholino) propanesulphonic A 0.15 0.18 for T. mentagrophytes) at 530 nm. Further
acid (MOPS) (Calbiochem, EMD Bioscience, La Jolla, CA). dilutions using sterile saline solution (0.9% NaCl) were
performed to obtain the final working inoculum concentration
Fungal strains tested (15 103 CFU/mL for Candida spp. and I. orientalis;
15 104 CFU/mL for C. neoformans; 0.45 104 CFU/mL
Candida albicans (ATCC 90028), Candida parapsilosis
for A. brasiliensis, and 1.26 104 CFU/mL for
(ATCC 22019), Issatchenkia orientalis (ATCC 6258),
T. mentagrophytes).
Cryptococcus neoformans (ATCC 90112), Aspergillus
brasiliensis (ATCC 16404), and Trichophyton mentagro-
Screening for antifungal activity
phytes (ATCC 9533) were purchased from the American
Type Culture Collection. The microorganisms were main- The colorimetric broth microdilution method of Eloff (1998)
tained on PDA at 4 C. was modified and employed for screening the extracts for
1528 P. S. Gwee et al. Pharm Biol, 2014; 52(12): 15261531
antifungal activity. This test utilized two-fold descending the sample (Perkin-Elmer Spectrum RX-1, Waltham, MA).
concentrations of the extracts in 96-well microplates and a HPTLC analysis and PLC separation were performed on silica
reference antibiotic, with concentrations ranging from 2.5 to gel plates (Kieselgel 60 F254, 0.2 mm; aluminum-backed and
0.02 mg/mL for the plant extracts, 0.25 to 0.002 mg/mL for 1 mm; glass-backed, respectively, Merck, Germany) using a
the fractions, 125 to 0.98 lg/mL for the isolated compounds, semi-automatic sample applicator (Camag Linomat IV,
and 8 to 0.063 lg/mL for amphotericin B. Growth, sterility, Switzerland). The HPTLC plate was scanned using a TLC
and negative controls for the extracts and medium were scanner (Camag TLC Scanner 3, Switzerland) and the data of
included. The 96-well microplates were incubated at 35 C for absorption signal spectra (200700 nm) were processed with
48 h for Candida spp. and I. orientalis; 72 h for C. neoformans WinCATS software (Planar Chromatography Manager version
and A. brasiliensis; and at 28 C for 7 d for T. mentagrophytes 1.4.4, Camag, Switzerland) for purity assessment. The HPTLC
(NCCLS, 2002a, 2002b). The colorimetric indicator, p- plates were photographed using a 12 bit charge-couple device
iodonitrotetrazolium violet (0.4 mg/mL), was added and a (CCD) digital camera (Camag TLC Visualizer, Switzerland)
color change from colorless to red indicated a positive result. under uniform illumination with white and UV light.
The concentration at which the color remains clear was The mass spectrum of the isolated compound was obtained
recorded as the MIC value. The MFC was obtained by by GC-MS (Shimadzu, QP2010 Plus, Tokyo, Japan) using
inoculating 20 lL of the preparation that showed no evidence helium as the carrier gas. The working pressure was 99.8 kPa
of growth during the MIC determination assays on PDA. and the flow rate was 1.46 mL/min. The GC-MS was fitted
The lowest concentration at which growth was not observed with a 5% phenyl polysilphenylene-siloxane (BPX5) capillary
was recorded as the MFC value. The tests were performed column of dimension 0.25 mm 30 m and the film thickness
in triplicate. was 0.25 lm. The injector port was maintained at 250 C
while the oven temperature gradient was 10 C/min from
Isolation of the active compound from N. gracilis 80 C to 310 C with a total program time of 37 min. Electron
impact ionization method was used. The ion source and
The most active extract, the hexane extract, was subjected to
interface temperatures were set at 200 C and 310 C,
liquidliquid partitioning. The dry hexane extract (1.12 g) was
respectively. The constituent was identified by comparing
dissolved in 150 mL of hexane. This was partitioned with an
with the mass spectral libraries (Wiley MS/NIST 05).
equal volume of water. The water layer was then extracted 1
H-NMR (400 MHz) and 13C-NMR (100 MHz) spectra of
using 150 mL of ethyl acetate. Each extraction was performed
the isolated compound were recorded in deuterium oxide
three-times and the extracts were pooled together. The hexane
(D2O) on a NMR instrument (JMN-ECX 400, JEOL, Tokyo,
fraction and the ethyl acetate fraction were evaporated to
Japan). All chemical shifts () were stated in parts per
dryness by rotary evaporation, while the water fraction was
million (ppm) with reference to tetramethylsilane (TMS) as
lyophilized. This yielded three fractions, i.e., hexane fraction
the internal standard and coupling constants (J) were
(F1), ethyl acetate fraction (F2), and water fraction (F3).
measured in Hz.
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 Determination of cytotoxic activity
strongest antifungal activity, was further purified by prepara-
LLC-MK2 cells (3 104 cells/well) were seeded in 96-well
tive thin layer chromatography (PLC) using a hexaneethyl
microplates and incubated for 24 h at 37 C in a humidified
acetate mixture (9:1, v/v) as the mobile phase. The F1 fraction
atmosphere with 5% CO2. After incubation, the cells were
was re-dissolved in hexane (2 mg/mL) and applied (190 mL) as
treated with two-fold serially diluted concentrations of
a thin even layer horizontally above the solvent level on a
plumbagin (0.056.25 lg/mL) for 72 h. The final concentra-
10 10 cm PLC plate. The development of PLC was
tion of the diluent (methanolwater mixture, 2:1, v/v) for
performed at room temperature. Two bands (assigned as B1
plumbagin was applied at the non-toxic concentration
and B2) were removed from the packing material (silica gel 60
(50.25%, v/v). Cell growth, sterility, and negative controls
F254) of the PLC plate. The silica gels from those bands were
were also included. The cell viability was evaluated using the
extracted twice with distilled water, followed by partitioning
NRU assay according to the protocol of Repetto et al. (2008)
with equal volume of hexane. The hexane solvent was
with modifications. The absorbance of the extracted neutral
collected and evaporated using a rotary evaporator and
red dye was measured at 540 nm using a microplate reader
screened for antifungal activity. Following recovery of
(Tecan Infinite-M200, Mannedorf, Switzerland). The experi-
components in B1, further purification was performed by re-
ment was performed in triplicate. The 50% cytotoxicity
crystallization in hexane. This yielded yellow crystals, which
concentration (CC50) of plumbagin on the cells was
was identified as 5-hydroxy-2-methyl-naphthalene-1,4-dione
determined from the plot of cell viability against concentra-
(plumbagin) based on spectroscopic data as well as by
tions of plumbagin. The data were analyzed with one-way
comparison with the literature data.
ANOVA using Statistical Package for the Social Sciences
(SPSS) software (Version 15.0 for Windows, SPSS Inc.,
Characterization of the active compound from
Chicago, IL) and the significance level was set at p50.05.
N. gracilis
The toxicity of plumbagin to the LLC-MK2 cells and the
Melting point was measured in a glass capillary tube by using antifungal activity of plumbagin against each species of
a melting point apparatus (Stuart SMP 10, Staffordshire, UK). fungus were compared by using the selectivity index (SI)
FT-IR spectroscopy was performed on KBr pellets containing 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.
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.
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.
The 1H- and 13C-NMR spectra confirmed the presence of activity of plumbagin isolated from the stem bark of
plumbagin with the molecular formula C11H8O3. Diospyros crassiflora. In this study, the concentration of
The viability of LLC-MK2 cells treated with various plumbagin that inhibited the growth of I. orientalis, and
concentrations of plumbagin as measured by NRU assay is T. mentagrophytes was about 2-fold and 4-fold lower,
given in Figure 2. Plumbagin was found to be significantly respectively, than that required for amphotericin B (Table 1).
cytotoxic (p50.05) to the cells at concentrations ranging The values of the selectivity indices of the isolated
from 0.78 to 6.25 lg/mL with the cell viability values less plumbagin against all the fungi were less than 1.0, indicating
than 10% after 72 h treatment. According to Figure 2, plumbagin is more toxic to mammalian than fungal cells.
the CC50 value of plumbagin on the LLC-MK2 cells was Seshadri et al. (2011) have shown that the cytotoxic effect of
0.60 mg/mL (3.19 lM). The cytotoxicity was not observed at plumbagin in human peripheral blood lymphocytes was at
concentrations below 0.20 lg/mL as the percentages of cell least two-fold higher than that of juglone (5-hydroxy-1,4-
viability were approximately 100%. The SI values calculated naphthalenedione). Plumbagin at 5 mM was proven to be non-
were 0.30 for C. albicans, I. orientalis, and T. mentagro- cytotoxic to the resting mouse lymphocytes, but at the
phytes, 0.15 for C. neoformans, 0.08 for C. parapsilosis and concentration of 2 mM was found to induce apoptosis in
0.02 for A. brasiliensis. human resting lymphocytes, indicating the species-specific
differences in the activity of plumbagin (Checker et al.,
2009). Two major mechanisms have been proposed for
Discussion
plumbagin cytotoxic action in various biological systems.
Several phenolic constituents, such as plumbagin, isoshina- First mechanism is associated with the excessive generation
nolone, epishinanolone, shinanolone, quercetin, kaempferol, of reactive oxygen species (ROS) such as superoxide radicals,
and flavonoid gallate esters, have been isolated from the singlet oxygen, and hydrogen peroxide (Castro et al., 2008;
leaves of N. gracilis (Aung et al., 2002; Fan et al., 2010). Seung et al., 1998). Second mechanism involved the redox
However, reports of the pharmacological and biological and oxidation cycle of quinones namely redox cycles
investigation of N. gracilis are scarce. To the best of our which lead to their ability to act as potent electrophiles to
knowledge, the present study is the first report to investigate inhibit the electron transportation, as uncouplers of oxidative
the antifungal property of N. gracilis leaf extracts against phosphorylation, as intercalating agent in the DNA double
fungi of medical important. helix and as bio-reductive alkylating agents in the biomol-
The six extracts obtained from the leaves of N. gracilis ecules (Babula et al., 2009). The redox cycling and free
exhibited antifungal activity against at least three types of radical forming was identified as the main mode of actions
fungi (Table 1). The bioactivity-guided approach led to the for plumbagin.
isolation of plumbagin from the hexane extract of N. gracilis. In animal studies, it has been noted that treatment with
Plumbagin is a secondary metabolite belonging to the plumbagin causes reproductive toxicity (Bhargava, 1984;
naphthoquinone group which is produced by many plants Premakumari et al., 1977). The oral bioavailability of
belonging to the Caryophyllales order (Richer et al., 2002) plumbagin in rat was less than 40%, due to its high
such as Droseraceae (Budzianowski, 2000), Plumbaginaceae lipophilicity and insolubility in water (Hsieh et al., 2006).
(Bothiraja et al., 2011), Ebenaceae (Evans et al., 1999), and Consequently, a large and frequent dose is necessary to
Nepenthaceae (Aung et al., 2002). A wide range of biological achieve the optimum therapeutic efficacy and this may lead to
properties of plumbagin such as anticancer (Kawiak et al., severe side effects. The toxicity of plumbagin is a major
2007), antiviral (Perez-Sacau et al., 2003), anti-inflamma- obstacle to its clinical application. The data reported herein
tory, antiplatelet, antiallergic (Lien et al., 1996), antimalarial are important, taking into account the medical importance of
(Biot et al., 2004), and antibacterial (Cai et al., 2000) have the studied fungi and that this plant species is traditionally
been reported. Dzoyem et al. (2007) reported the antifungal consumed or applied externally, without considering the
DOI: 10.3109/13880209.2014.902083 Antifungal activity of Nepenthes gracilis 1531
presence of toxic components such as plumbagin. Thus, the Eloff JN. (1998). A sensitive and quick microplate method to determine
the minimal inhibitory concentration of plant extracts for bacteria.
pharmacological study of medicinal plants remains important Planta Med 64:71114.
to provide evidence with scientific basis for the continued Evans PH, Bowers WS, Litaudon M, Sevenet T. (1999). Plumbagin from
traditional application of plants and understanding of a plants Diospyros olen. Molecules 4:M93.
efficacy as well as toxicity. Fan DH, Wang H, Zhi D, Shen YM. (2010). CE analysis of endogenous
flavonoid gallate esters from Nepenthes gracilis (Nepenthaceae).
Chromatographia 72:101316.
Conclusions Heitman J, Filler SG, Edwards Jr JE, Mitchell AP. (2006). Molecular
Principles of Fungal Pathogenesis. Washington, USA: ASM Press,
Plumbagin plays a significant role in the antifungal activity of 311.
N. gracilis. The present study demonstrated that the leaves of Hsieh YJ, Lin LC, Tsai TH. (2006). Measurement and pharmacokinetic
this plant could serve as an antifungal agent but caution study of plumbagin in a conscious freely moving rat using liquid
chromatography/tandem mass spectrometry. J Chromatogr B Analyt
should be taken as plumbagin is a potentially cytotoxic Technol Biomed Life Sci 844:15.
compound. 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.
Acknowledgements Kriengkauykiat J, Ito JI, Dadwal SS. (2011). Epidemiology and
The authors thank Universiti Tunku Abdul Rahman for a treatment approaches in management of invasive fungal infections.
Clin Epidemiol 3:17591.
research grant (UTARRF Vote No. 6200/S07) which sup- Lien J, Huang L, Wang J, et al. (1996). Synthesis and antiplatelet, anti-
ported the M.Sc. candidature of Pei Shing Gwee. 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://
Declaration of interest onlinelibrary.wiley.com/doi/10.1111/j.1439-0507.2011.02032.x/pdf
[last accessed 9 Feb 2012].
The authors declare no conflict of interest. National Committee on Clinical Laboratory Standards (2002a).
Reference Method for Broth Dilution Antifungal Susceptibility
References Testing of Yeasts; Approved Standard, NCCLS Document M27-A2,
2nd ed. Pennsylvania: NCCLS.
Aung HH, Chia LS, Goh NK, et al. (2002). Phenolic constituents from National Committee on Clinical Laboratory Standards (2002b).
the leaves of the carnivorous plant Nepenthes gracilis. Fitoterapia 73: Reference Method for Broth Dilution Antifungal Susceptibility
4457. Testing of Filamentous Fungi; Approved Standard, NCCLS
Babula P, Adam V, Havel L, Kizek R. (2009). Noteworthy secondary Document M38-A. Pennsylvania: NCCLS.
metabolites napthoquinones their occurrence, pharmacological Okeke IN, Laxmaninarayan R, Bhutta ZA, et al. (2005). Antimicrobial
properties and analysis. Curr Pharm Anal 5:4768. resistance in developing countries. Part 1. Recent trends and current
Bhargava SK. (1984). Effects of plumbagin on reproductive function of status. Lancet Infect Dis 12:48193.
male dog. Ind J Exp Biol 22:1536. Pappas PG, Alexander BD, Andes DR, et al. (2010). Invasive fungal
Biot C, Bauer H, Schirmer R, Davioud-Charvet E. (2004). 5-Substituted infections among organ transplant recipients: Results of the
tetrazoles as bioisosteres of carboxylic acids. Bioisosterism and Transplant-Associated Infection Surveillance Network
mechanistic studies on glutathione reductase inhibitors as antimalar- (TRANSNET). Clin Infect Dis 50:110111.
ials. J Med Chem 47:597283. Perez-Sacau E, Estevez-Braun A, Ravelo A, et al. (2003). Inhibitory
Bothiraja C, Joshi PP, Dama GY, Pawar AP. (2011). Rapid method for effects of lapachol derivatives on Epstein-barr virus activation. Bioorg
isolation of plumbagin, an alternative medicine from roots of Med Chem 11:4838.
Plumbago zeylanica. Eur J Integrative Med 3:3942. Premakumari P, Rathinam K, Santhakumari G. (1977). Antifertility
Budzianowski J. (2000). Napthoquinone glucosides of Drosera gigantea activity of plumbagin. Ind J Med Res 65:82938.
from in vitro cultures. Planta Med 66:6679. Repetto G, del Peso A, Zurita JL. (2008). Neutral red uptake assay for the
Burkill IH. (1966). A Dictionary of the Economic Products of Malay estimation of cell viability/cytotoxicity. Nat Protoc 3:112531.
Peninsula, Vol. 2. Kuala Lumpur: Ministry of Agriculture and Richer H, Hamm A, Bringmann G. (2002). Nepenthes insignis uses a C-
Cooperatives. 2-portion of the carbon skeleton of L-alanine acquired via its
Cai L, Wei G-X, Van der Bijl P, Wu CD. (2000). Namibian chewing carnivores organs, to build up the allelochemical plumbagin.
stick, Diospyros lycioides, contain antibacterial compounds against Phytochemistry 59:6039.
oral pathogens. J Agric Food Chem 48:90914. Seshadri P, Rajaram A, Rajaram R. (2011). Plumbagin and juglone
Castro FAV, Mariani D, Panek AD, et al. (2008). Cytotoxicity induce caspase-3-dependent apoptosis involving the mitochondria
mechanism of two naphthoquinones (menadione and plumbagin) in through ROS generation in human peripheral blood lymphocytes. Free
Saccharomyces cerevisiae. PLoS One [Online]. Available from: http:// Radic Biol Med 51:2090107.
www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone. Seung S, Lee JY, Lee MY, et al. (1998). The relative importance of
0003999#pone-0003999-g003 [last accessed 5 Jan 2014]. oxidative stress versus arylation in the mechanism of quinone-induced
Checker R, Sharma D, Sandur SK, et al. (2009). Anti-inflammatory cytotoxicity to platelets. Chem Biol Interact 113:13344.
effects of plumbagin are mediated by inhibition of NF-kappaB Shin KS, Lee S, Cha BJ. (2007a). Suppression of phytopathogenic fungi
activation in lymphocytes. Int Immunopharmacol 9:94958. by hexane extract of Nepenthes ventricosa x maxima leaf. Fitoterapia
Denning DW, Hope WW. (2010). Therapy for fungal diseases: 78:5856.
Opportunities and priorities. Trends Microbiol 18:195204. Shin KS, Lee S, Cha BJ. (2007b). Antifungal activity of plumbagin
Dzoyem JP, Tangmouo JG, Lontsi D, et al. (2007). In vitro antifungal purified from leaves of Nepenthes ventricosa x maxima against
activity of extract and plumbagin from the stem bark of Diospyros phytopathogenic fungi. Plant Pathol J 23:11315.
crassiflora Hiern (Ebenaceae). Phytother Res 21:6714. Wiart C, Mogana S, Khalifah S, et al. (2004). Antimicrobial screening of
Elison AM, Gotelli NJ. (2001). Evolutionary ecology of carnivorous plants used for traditional medicine in the state of Perak, Peninsular
plants. Trends Ecol Evol 16:6239. Malaysia. Fitoterapia 75:6873.