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Antifungal activity of Andrographis paniculata extracts and active principles against skin pathogenic fungal strains in vitro

Authors:
  • Universiti Kebangsaan Malaysia Bandar Baru Bangi

Abstract and Figures

Andrographis paniculata Nees. (Acanthaceae) is an annual herbaceous plant widely cultivated in southern Asia, China, and Europe. It is used in the treatment of skin infections in India, China, and Malaysia by folk medicine practitioners. Antifungal activity of the whole plant extracts and isolation of active principles from A. paniculata were investigated. Dichloromethane (DCM) and methanol (MEOH) extracts of A. paniculata whole plant were screened for their antifungal potential using broth microdilution method in vitro against seven pathogenic fungal species responsible for skin infections. Active principles were detected through bioguided assays and isolated using chromatography techniques. Structures of compounds were elucidated through spectroscopy techniques and comparisons were made with previously reported data for similar compounds. DCM extract revealed lowest minimum inhibitory concentration (MIC) value (100 μg/mL) against Microsporum canis, Candida albicans, and Candida tropicalis, whereas MEOH extract revealed lowest MIC (150 µg/mL) against C. tropicalis and Aspergillus niger. DCM extract showed lowest minimum fungicidal concentration (MFC) value (250 µg/mL) against M. canis, C. albicans, C. tropicalis and A. niger, whereas MEOH extract showed lowest MFC (250 µg/mL) against Trichophyton mentagrophytes, Trichophyton rubrum, M. canis, C. albicans, C. tropicalis and A. niger. Bioassay guided isolation from DCM and MEOH extract afforded 3-O-β-d-glucosyl-14-deoxyandrographiside, 14-deoxyandrographolide, and 14-deoxy-11,12-didehydroandrographolide as antifungal compounds. The lowest MIC (50 µg/mL) and MFC (50 µg/mL) was exerted by 14-deoxyandrographolide on M. canis. This is first report on the isolation of antifungal substances through bioassay-guided assay from A. paniculata. Our finding justifies the use of A. paniculata in folk medicines for the treatment of fungal skin infections.
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850
Introduction
Infectious diseases are one of the main causes of mor-
bidity and mortality worldwide (Meena et al., 2010).
During the past several years, there has been an increas-
ing incidence of fungal infections due to an increase
in immune-compromised population such as organ
transplant recipients, cancer, and HIV/AIDS patients.
Fungal infections though not as frequent as bacterial
or viral infections, have nonetheless been increasing in
incidence in the human population over the last 20 years.
is fact coupled with the resistance to antibiotics and
with the toxicity during prolonged treatment with several
RESEARCH ARTICLE
Antifungal activity of Andrographis paniculata extracts and
active principles against skin pathogenic fungal strains in vitro
Abubakar Sule1, Qamar Uddin Ahmed2, Jalifah Latip3, Othman Abd. Samah1,
Muhammad Nor Omar1, Abdulrashid Umar2, and Bashar Bello S. Dogarai2
1Department of Biomedical Sciences, Faculty of Science, 2Department of Pharmaceutical Chemistry, Faculty of
Pharmacy, International Islamic University Malaysia (IIUM), Kuantan, Pahang Darul Makmur, Malaysia, and
3School of Chemical Sciences & Food Technology, Faculty of Science & Technology, University Kebangsaan Malaysia,
UKM Bangi, Selangor Malaysia
Abstract
Context: Andrographis paniculata Nees. (Acanthaceae) is an annual herbaceous plant widely cultivated in southern
Asia, China, and Europe. It is used in the treatment of skin infections in India, China, and Malaysia by folk medicine
practitioners.
Objective: Antifungal activity of the whole plant extracts and isolation of active principles from A. paniculata were
investigated.
Materials and methods: Dichloromethane (DCM) and methanol (MEOH) extracts of A. paniculata whole plant were
screened for their antifungal potential using broth microdilution method in vitro against seven pathogenic fungal
species responsible for skin infections. Active principles were detected through bioguided assays and isolated using
chromatography techniques. Structures of compounds were elucidated through spectroscopy techniques and
comparisons were made with previously reported data for similar compounds.
Results: DCM extract revealed lowest minimum inhibitory concentration (MIC) value (100 μg/mL) against Microsporum
canis, Candida albicans, and Candida tropicalis, whereas MEOH extract revealed lowest MIC (150 µg/mL) against C.
tropicalis and Aspergillus niger. DCM extract showed lowest minimum fungicidal concentration (MFC) value (250
µg/mL) against M. canis, C. albicans, C. tropicalis and A. niger, whereas MEOH extract showed lowest MFC (250 µg/
mL) against Trichophyton mentagrophytes, Trichophyton rubrum, M. canis, C. albicans, C. tropicalis and A. niger.
Bioassay guided isolation from DCM and MEOH extract aorded 3-O-β-D-glucosyl-14-deoxyandrographiside,
14-deoxyandrographolide, and 14-deoxy-11,12-didehydroandrographolide as antifungal compounds. The lowest
MIC (50 µg/mL) and MFC (50 µg/mL) was exerted by 14-deoxyandrographolide on M. canis.
Discussion and conclusion: This is rst report on the isolation of antifungal substances through bioassay-guided assay
from A. paniculata. Our nding justies the use of A. paniculata in folk medicines for the treatment of fungal skin
infections.
Keywords: Andrographis paniculata, antifungal activity, active principles, skin infections, dermatology, MIC, MFC
Address for Correspondence: Dr. Qamar U. Ahmed, Department of Pharmaceutical Chemistry, Faculty of Pharmacy, International
Islamic University Malaysia (IIUM), 25200 Kuantan, Pahang Darul Makmur, Malaysia. Tel: 006-09-5716400 (3096). Fax: 006-09-571 6775.
E-mail: quahmed@iium.edu.my, qamaruahmed@yahoo.com
(Received 03 December 2010; revised 04 November 2011; accepted 11 November 2011)
Pharmaceutical Biology, 2012; 50(7): 850–856
© 2012 Informa Healthcare USA, Inc.
ISSN 1388-0209 print/ISSN 1744-5116 online
DOI: 10.3109/13880209.2011.641021
Pharmaceutical Biology
2012
50
7
850
856
03 December 2010
04 November 2011
11 November 2011
1388-0209
1744-5116
© 2012 Informa Healthcare USA, Inc.
10.3109/13880209.2011.641021
NPHB
641021
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Antifungal activity of Andrographis paniculata 851
© 2012 Informa Healthcare USA, Inc.
antifungal drugs, is the reason for an extended search for
new drugs to treat opportunistic fungal infections (Fostel
& Lartey, 2000). Hence, eective antifungal therapy could
prove a signicant role in health care; the screening of
traditional medicinal plants in search of novel antifungal
agents is now more frequently performed. e search for
novel antifungal agents relies greatly on ethnobotanical
information and ethno-pharmacological exploration
(Motsei et al., 2003; Fortes et al., 2008).
Andrographis paniculata (Burm.f.) Wall. ex Nees.,
(Acanthaceae) (King of Bitters, Hempedu Bumi in Malay)
is an annual herbaceous plant widely cultivated in south-
ern Asia, China, and some parts of Europe. e whole
plant and roots have traditionally been used over the
centuries in Asia and Europe as a folk medicine for a wide
variety of ailments or as herbal supplements for health
promotion (Chopra et al., 1982; Khory & Katrak, 1984;
Chang & But, 1987). In traditional Chinese medicine, it
is widely used to get rid of body heat, as in fevers, and to
dispel toxins from the body (Tang & Eisenbrand, 1992).
In European countries, it is frequently used to prevent
the common cold (Caceras et al., 1997). A. paniculata
has been reported to have a wide range of pharmacologi-
cal activities antidiabetic (Syahrin et al., 2006), antivi-
ral (Wiart et al., 2005), antibacterial (Singha et al., 2003;
Sule et al., 2011a, 2011b), anticancer (Tan et al., 2005;
Geethangili et al., 2008), antiinammatory (Shen et al.,
2002), hepatoprotective (Trivedi & Rawal, 2005), immune-
stimulatory/immunomodulatory (Iruretagoyena et al.,
2005; Wang et al., 2010), and antisnakebite activity (Samy
et al., 2008).
Evidence of its wide use by the traditional clerics in
India, Malaysia, and China for treating some skin infec-
tions (Tapsell et al., 2006) and its promising in vitro
antibacterial activity (Singha et al., 2003; Sule et al.,
2011a,b) prompted us to choose this plant for further
evaluation in order to ascertain its antifungal potential
and responsible agents to treat skin infections that are
caused by pathogenic fungi. is area of the pharmaco-
logical activity of this plant has not yet been thoroughly
investigated.
Materials and methods
Instruments
Melting points of all compounds were determined
using Buchi B-545 instrument (Buchi, Switzerland).
UV spectra were determined on SECOMAM UV-Vis
Spectrophotometer (Jena Analytic, Germany). IR spec-
tra were recorded on Perkin Elmer Spectrum RXI FT-IR
Spectrometer using KBr disks. 1H- and 13C-NMR spectra
were recorded on a Bruker instrument at 600 MHz and
150 MHz, respectively.
Collection of plant material
Fresh whole plant of A. paniculata was procured from the
botanical garden of Forest Research Institute of Malaysia
(FRIM), Kuala Lumpur, Malaysia, during the month
of April, 2009. e plant was identied by Dr. Richard
Chung Cheng Kong (Taxonomist), FRIM, Malaysia. e
voucher specimen (NMPC-Q25) has been deposited in
the Herbarium, Faculty of Pharmacy, IIUM, Kuantan,
Pahang DM, Malaysia for future references.
Preparation of dichloromethane (DCM) and methanol
(MEOH) extracts
e fresh A. paniculata whole plant (5 kg) was dried in
a PROTECH laboratory air dryer (FDD-720-Malaysia)
at 40°C for 7 days and pulverized [600 g (12%)] using
Fritsch Universal Cutting Mill-PULVERISETTE 19-Ger-
many. It was then stored in a desiccator at 2°C until
further use. All solvents were double distilled before
use. Dry powdered A. paniculata whole plant was suc-
cessively extracted with DCM and MEOH using Soxhlet
apparatus for 6 h separately. e extracts were ltered
and concentrated using a rotary evaporator (Buchi
Rotary Evaporator, R-210, Switzerland). Final concen-
trated extracts upon freeze drying [DCM extract 10.10 g
(4.04%) and MEOH extract 19.25 g (7.07%)] were stored
at 2°C in labeled sterile bottles and kept as aliquots until
further antifungal evaluation. All chemicals used in this
study were of analytical grade and double distilled.
Test microorganisms
Seven reference fungal strains notable for skin patho-
genesis were chosen for antifungal investigation:
three dermatophytes [Trich ophyton men tagr ophytes
(IMR T-44), Trichophyto n rubr um (ATCC-28188),
Microsporum canis (ATCC-36299)], one non-dermato-
phyte [Aspergillus niger (IMR A-102)], and three yeasts
[Candida albicans (ATCC-90028), Candida krusei (IMR
C-368), Candida tropicalis (IMR C-353)]. All fungal
strains were purchased either from the Institute for
Medical Research (IMR), Malaysia or from American
Type Culture Collection (ATCC), USA, respectively. All
fungal stock cultures were maintained on Potato dex-
trose agar (PDA) (for non-dermatophyte and yeasts)
and Sabouraud dextrose agar (SDA) (for dermatophytes)
slants (OXOID Ltd., England) at 4°C and pH 5.6.
Screening for antifungal activity
Determination of minimum inhibitory concentration (MIC)
and minimum fungicidal concentration (MFC)
Broth microdilution method recommended by Clinical
and Laboratory Standards Institute, USA (CLSI, 2008a,b)
was used for the determination of MIC and MFC values
for each plant extract and bioguided isolated com-
pounds. A two-fold dilution series of plant extracts (DCM
and MEOH) and compounds dissolved in 10% DMSO
(which had no inhibitory activity against test micro-
organisms) was prepared: 400, 350, 300, 250, 200, 150,
100, 50 and 01 µg/mL, this was then added to the broth
media in a 96-wells microtiter plates. Subsequently, 100
µL of inoculum (adjusted to 107 CFU/mL) was added
to each well. Fungal suspensions with 10% DMSO was
used as negative control, whereas broth containing
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852 A. Sule et al.
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standard drugs, nystatin (NY) and griseofulvin (GR) (30
µg/mL) were used as positive controls. Each extract was
assayed in duplicate and each time two sets of microti-
ter plates were prepared, one was kept for incubation
while another set was kept at 4°C for comparing the tur-
bidity in the wells of microtitre plate. MIC values were
determined as the lowest concentration of the extracts/
compounds that showed no turbidity after incubation.
e turbidity of the wells in the microtiter plate was
interpreted as visible growth of microorganisms. e
MFC was determined by subculturing 50 µL from each
well showing no apparent growth onto freshly prepared
SDA plates incubated at 32°C for 48 h (dermatophytes)
and PDA plates incubated at 28°C for 48 h (yeast and
non-dermatophyte). Lowest concentration of extract/
compounds showing no visible growth on subculturing
was taken as MFC.
Bioautography
MEOH and DCM (10 µL each) extracts were applied as
small spots separately on sterilized 10 × 10 cm TLC plates
and developed in hexane: acetone (2:1). Broth cultures of
A. niger, C. tropicalis and M. canis (adjusted to 107 CFU/
mL) were mixed with 30 mL molten SDA separately.
Mixture of agar and microbial suspensions were spread
aseptically onto the TLC plates in square Petri dishes,
allowed for 25 min to solidify, and then the plates were
incubated at 32°C for 48 h. At the end of incubation time,
0.5% p-iodonitrotetrazolium violet (INT) was uniformly
sprayed on the TLC plates and the active antifungal com-
pounds in the plant extracts were detected as whitish
TLC spots on a deep pink background after incubation
in the moist atmosphere for 2–3 days at 25°C (Rahalison
et al., 2007).
Bioguided isolation of active principles
e air dried and powdered whole plant (5 kg) of A.
paniculata was extracted by macerating in methanol
(20.0 L) at room temperature for 24 h, ltered, and
evaporated under reduced pressure. e whole process
was repeated three times to ensure maximum yield of
methanol soluble compounds from the plant powder.
Each time, ltrate was evaporated under reduced pres-
sure and combined. e dark blackish green residue was
further extracted with DCM and MEOH, respectively, in
order to get DCM and MEOH soluble compounds sepa-
rately. e DCM soluble portion upon evaporation under
reduced pressure yielded 120 g DCM extract and MEOH
soluble portion yielded 217 g MEOH extract. DCM and
MEOH extracts were chromatographed separately on
a silica gel column using dierent polarity to obtain
several fractions. ese fractions were further chro-
matographed on a silica gel column to aord three anti-
microbial compounds viz., AB-1 (22 mg), AB-2 (25 mg),
and AB-3 (13 mg). e structures of isolated antifungal
constituents were unambiguously elucidated based on
chemical evidences, spectral analysis and comparison
with already reported data.
Identification and isolation of antifungal compounds
(AB-1 and AB-2) from MEOH extract
MEOH extract (100 g) was loaded onto column
(10 × 50 cm) packed with silica gel 60 particle size
0.063–0.2 mm (70–230 mesh) (Fluka Chemika). e
column was eluted with pure hexane (100%) through
hexane:ethyl acetate with increasing polarity (90:10,
80:20, 70:30, 50:50, 30:70, 20:80, 10:90, 0:100) and
nally with ethyl acetate:methanol with increasing
polarity (95:5, 90:10, 80:20, 70:30, 50:50, 30:70, 20:80,
0:100); 50 mL were collected in individual test tubes,
214 fractions were obtained and eluents from test
tubes that exhibited similar R.f. values as indicated by
TLC analysis in dierent solvent systems were pooled
together and a total of 20 (AM–TM) similar fractions were
eventually obtained. Antifungal active fractions IM–LM
and NM–OM aorded crystallized products of two com-
pounds which were further puried using preparative
column chromatography on silica gel 60 and eluted
with hexane:ethyl acetate to produce various fractions.
Eluents in test tubes 78–85, 88–97, 98–107 and 108–
122 upon crystallization with absolute ethyl alcohol
aorded pure compound AB-1 (white crystals) (22 mg);
M.P. 242–244°C; R.f. 0.78 (chloroform:methanol:ethyla
cetate (CME) (16: 0.8: 1.2)); UV λmax MeOH nm: 202; IR
(cm1) ν: 3351, 1732, 165, 899 and AB-2 (colorless crys-
tals) (25 mg); M.P. 172–174°C, R.f. 0.66 (CME); UV λmax
MeOH nm: 223 and IR (cm1) ν: 3367, 1736, 1646, 896.
1H-NMR and 13C-NMR spectral data of AB-1 and AB-2
are given in Table 1.
Identification and isolation of antifungal compound
(AB-3) from DCM extracts
DCM extract (100 g) was loaded onto column (10 × 50 cm)
packed with silica gel 60 particle size 0.063–0.2 mm (70–230
mesh) (Fluka Chemika). e column was eluted with
750 mL at a gradient system beginning with pure hexane
(100%) through hexane:acetone with increasing polarity
(90:10, 80:20, 70:30, 50:50, 30:70, 20:80, 10:90, 0:100) and
nally with acetone:methanol with increasing polarity
(95:5, 90:10, 80:20, 70:30, 50:50, 30:70, 20:80, 0:100). 50 mL
were collected in test tubes, 145 fractions were obtained
and eluents from test tubes that exhibited similar R.f. val-
ues pooled together and a total of 13 (AD–MD) similar frac-
tions were eventually obtained. ese were put in 25 mL
conical ask in ethanol for crystallization and recrystal-
lization to obtain pure compounds. Antifungal active
fractions FD–ID and JD–MD were pooled together based on
TLC prole and kept for recrytallization to aord the mix-
ture of three crystallized substances which were further
puried by using preparative column chromatography
on silica gel 60 and eluted with hexane:ethylacetate to
produce various fractions. Eluents in test tubes 32–36,
62, 101–106, 107–112 and 113–145 upon recrystallization
aorded compound AB-3 (whitish crystals) (13 mg); M.P.
208–209°C; R.f. 0.45 (CME); UV λmax MeOH nm: 209 and
IR (cm1) ν: 3373, 1741, 1642, 898. 1H-NMR an d 13C-NMR
spectral data of AB-3 are given in Ta b le 1.
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Antifungal activity of Andrographis paniculata 853
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Results
Results of our study showed that the whole plant extracts
have good inhibitory eects against most of the pathogenic
fungal species taken into consideration. MIC and MFC of
active extracts were determined by broth microdilution
assay. Highest MIC value was exerted by DCM extract
at 250 µg/mL on C. krusei, A. niger, and the lowest was
exerted at 100 µg/mL on M. canis, C. albicans, C. tropicalis,
whereas MEOH extract revealed highest MIC (200 µg/mL)
against T. mentagrophytes, T. rubrum, M. canis, C. albicans,
C. krusei and showed lowest MIC (150 µg/mL) against C.
tropicalis and A. niger. DCM extract showed highest MFC
(300 µg/mL) against T. mentagrophytes, T. rubrum, C.
krusei, and lowest MFC (250 µg/mL) against M. canis, C.
albicans, C. tropicalis and A. niger. MEOH extract showed
highest MFC (350 µg/mL) on C. krusei and the lowest MFC
(250 µg/mL) T. mentagrophytes, T. rubrum, M. canis, C.
albicans, C. tropicalis and A. niger (Ta b l e 2).
Bioguided isolation from DCM and MEOH extracts
of the whole plant of A. paniculata aorded three
antifungal compounds viz., 3-O-β-d-glucosyl-14-
deoxyandrographiside (AB-1), 14-deoxyandrographolide
(AB-2) and 14-deoxy-11,12-dihydrondrographolide
(AB-3). eir structures were unambiguously character-
ized through the analysis of UV, IR, 1H-NMR and 13C-NMR
spectral data and direct comparison with the previously
reported spectral data of similar compounds (Zhou et al.,
2008; Poonam et al., 2010).
Compound AB-1 obtained from MEOH extract showed
positive Legal and Kedde test, suggests the presence of
Table 2. Minimum inhibitory concentration (MIC) and
minimum fungicidal concentration (MFC) of DCM and MEOH
extracts of A. paniculata whole plant.
Fungal strains
Minimum
inhibitory
concentrations
(MIC) µg/mL
Minimum
fungicidal
concentration
(MFC) µg/mL
DCM MEOH DCM MEOH
Trichophyton mentagrophytes 200 200 300 250
Trichophyton rubrum 200 200 300 250
Microsporum canis 100 200 250 250
Candida albicans 100 200 250 250
Candida krusei 250 200 300 350
Candida tropicalis 100 150 250 250
Aspergillus niger 250 150 250 250
DCM, dichloromethane extract; MEOH, methanol extract.
Table 1. 1H- and 13C-NMR chemical shifts of AB-1 (3-O-β-d-glucosyl-14-deoxyandrographiside), AB-2 (14-deoxyandrographolide) and
AB-3 (14-deoxy-11,12-dihydrondrographolide), (chemical shifts in δ ppm), 600 and 150 MHz in CDCl3, “o” denotes overlapping signals, J
values are given in Hertz (Hz).
Position
AB-1 AB-2 AB-3
δHδCδHδCδHδC
1 1.71 (o, 1H), 1.25 (o, 1H) 38.19 1.32 (m, 2H) 38.93 1.53 (m, 1H), 1.12 (m, 1H) 38.59
2 2.10 (m, 1H), 1.98 (m, 1H) 29.72 2.04 (brs, 1H), 1.99 (o, 1H) 37.74 1.75 (m, 1H), 1.60 (m, 1H) 28.15
3 3.92 (o, 1H) 75.04 3.35 (o, 1H) 80.50 3.53 (m, 1H) 80.66
4 39.56 64.13 – 64.20
5 1.3 (m, 1H) 56.44 1.46 (o, 1H) 55.21 1.14 (m, 1H) 55.30
6 1.85 (m, 2H) 35.97 2.18 (brs, 1H), 1.98 (brd, J = 6, 1H) 28.22 1.81 (m, 2H) 28.28
7 2.4 (m, 2H) 38.44 2.45 (t, 2H) 37.06 2.03 (m, 1H), 1.94 (m, 1H) 38.04
8 147.30 148.94 – 148.10
9 3.35 (d, J = 8.4, 1H) 56.18 3.50 (o, 1H) 55.96 2.33 (d, J = 10.2, 1H) 56.04
10 38.89 42.96 – 42.95
11 1.8 (m, 2H) 21.72 2.43 (dd, J = 4.2, 2.4, 2H) 24.88 6.90 (dd, J = 15.6, 9.6, 1H) 134.71
12 2.6 (m, 1H), 2.3 (m, 1H) 24.46 2.55 (dd, J = 12, 7.2, 2H) 23.76 6.13 (d, J = 15.6, 1H) 121.12
13 136.02 146.64 – 146.79
14 7.10 (t, 1H) 143.84 7.10 (brs, 1H) 127.83 7.10 (t, 1H) 129.30
15 4.77 (brs, 2H) 70.11 4.91 (brs, 2H) 66.31 4.19 (d, J = 10.8, 2H) 69.63
16 174.33 172.17 – 172.29
17 4.87 (brs, 1H), 4.59 (brs, 1H) 107.08 4.59 (brs, 1H), 4.45 (brs, 1H) 108.86 4.89 (d, J = 1.8, 1H), 4.60
(brs, 1H)
109.2
18 1.00 (brs, 3H) 18.93 1.59 (o, 3H) 22.67 1.27 (s, 3H) 22.69
19 4.03 (d, J = 9.6, 1H), 3.22
(d, J = 9.6, 1H)
62.61 4.20 (brs, 1H), 4.18 (brs, 1H) 74.61 4.23 (d, J = 10.8, 1H), 3.50
(d, J = 10.8, 1H)
70.12
20 0.66 (brs, 3H) 15.41 0.71 (s, 3H) 15.16 0.82 (s, 3H) 15.91
14.24 (d, J = 7.2, 1H) 103.10
23.37 (o, 1H) 71.72
33.39 (o, 1H) 72.73
43.57 (o, 1H) 74.03
53.59 (o, 1H) 76.23
63.84 (dd, J = 11.4, 4.8, 2H) 70.72
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an α, β-unsaturated lactone in the compound. e 1H-
and 13C-NMR spectra of AB-1 revealed signals due to a
β-glucopyranosyl group [δH 4.24 (d, J = 7.2 Hz, 1H)] and
δC 103.10, 71.72, 72.73, 74.03, 76.23 and 70.72 and the
characteristic signals for the double bond containing one
hydrogen at carbon 14 in γ-lactone ring were observed at
δ 7.10 (t, 1H) in 1H-NMR as well as in 13C-NMR spectra at
δ 143.84, respectively (Table 1), which corresponds to the
3-O-β-d-glucosyl-14-deoxyandrographiside previously
isolated from A. paniculata and reported by Zhou et al.
(2008) (Figure 1a). Compound AB-2 was also obtained
from MEOH extract exhibited positive test for the Legal
and Kedde reactions, suggests the presence of an α,
β-unsaturated lactone in the molecule. e character-
istic NMR spectral data indicated that compound AB-2
was a labdane-type diterpene with α, β-unsaturated
γ-lactone. In 1H-NMR spectrum of AB-2, two methyl sin-
glets were observed at δ 0.71 and 1.59, respectively. e
characteristic exocyclic methylene protons for AB-2 diter-
penoids were observed at δ 4.59 (brs, 1H) and 4.45 (brs,
1H) in 1H-NMR as well as at δ 108.86 in 13C-NMR spectra
respectively (Table 1). e 1H- and 13C-NMR (in CDCl3)
spectra of AB-2 suggested a diterpenoid compound with
a structure similar to that of 14-deoxyandrographolide
previously isolated from A. paniculata by Poonam et al.
(2010) (Figure 1b). However, compound AB-3 was iso-
lated from the DCM extract and its characteristic NMR
spectral data indicated to be a labdane-type diterpene
with an α, β-unsaturated γ-lactone. AB-3 was also found
to be positive for the Legal and Kedde reactions, suggests
the presence of an α, β-unsaturated lactone in the same
molecule. In 1H-NMR spectrum, two methyl singlets were
observed at δ 0.82 and 1.27, respectively. e character-
istic exocyclic methylene protons for diterpenoids were
observed at δ 4.89 (d, J = 1.8, 1H) and 4.60 (brs, 1H) in
1H-NMR as well as at δ 109.2 in 13C-NMR spectra respec-
tively and the signals for the presence of one hydrogen
each residing at C-11 and C-12 were observed in the form
of doublet of doublets and single doublet at δ 6.90 (dd,
J = 15.6, 9.6 Hz, 1H) and 6.13 (d, J = 15.6 Hz, 1H) as well
as at δ 134.71 and 121.12 in 13C-NMR, respectively. e
characteristic signals for the double bond containing one
hydrogen at carbon 14 for diterpenoids were observed
at δ 7.10 (t, 1H) in 1H-NMR as well as in 13C-NMR at δ
129.30, respectively. Two signals in the form of doublet
of doublets and doublet for didehydro at carbon 11 and
carbon 12 in the 1H-NMR spectrum were observed at δ
6.90 (dd, J = 15.6, 9.6 Hz, 1H) and 6.13 (d, J = 15.6 Hz, 1H),
respectively which were also correlated to the signals at
δ 134.71 and 121.12 in 13C-NMR spectrum (Table 1). e
1H- and 13C-NMR (in CDCl3) spectra of AB-3 explicitly
suggested a diterpenoid compound with a structure sim-
ilar to that of 14-deoxy-11,12-didehydrondrographolide
previously isolated from A. paniculata and reported by
Poonam et al. (2010) (Figure 1c).
All three compounds were further investigated for
their thorough antifungal activity by broth microdilution
method against three fungal strains (M. canis, A. niger,
and C. albicans) on which the plant extracts revealed
the most potent antifungal activity. MIC values for all
antifungal compounds ranged from 50–150 µg/mL and
MFC values ranged from 50–200 µg/mL. e highest
MIC (150 µg/mL) was exerted by compound AB-2 and
AB-3 against C. tropicalis and M. canis, whereas AB-2
revealed the lowest MIC (50 µg/mL) indicates the most
potent antifungal activity against M. canis. e high-
est MFC (200 µg/mL) was exerted by compound AB-2
on C. tropicalis and the lowest MFC (50 µg/mL) was
exerted by compound AB-2 on M. canis. Compound
AB-3 (14-deoxy-11,12-didehydrondrographolide)
showed broad spectrum antifungal activity as it was
found to exhibit inhibitory activity against all fungal
strains taken into consideration. In contrast, positive
control, nystatin showed the lowest MIC (5 µg/mL) and
MFC (10 µg/mL) on M. canis, and griseofulvin revealed
the lowest MIC (10 µg/mL) and MFC (20 µg/mL) on
Figure 1. (a) Structure of AB-1 (3-O-β-d-glucosyl-14-deoxyan-
drographiside) obtained from MEOH extract of A. paniculata whole
plant.(b) Structure of AB-2 (14-deoxyandrographolide) obtained
from MEOH extract of A. paniculata whole plant. (c) Structure of
AB-3 (14-deoxy-11,12-didehydrondrographolide) obtained from
DCM extract of A. paniculata whole plant.
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A. niger (Tab l e 3). e results clearly revealed that all
compounds including standard antibiotics were selec-
tive in their mode of action on the tested strains. As
observed from the results, the antifungal activities of
the compounds were more prominent on dermato-
phytes followed by non-dermatophyte and yeast strains
tested. e isolated compounds demonstrated their
antifungal eect (high MIC and MFC values) though to
a lesser extent as compared to the positive controls i.e.,
standard antibiotics.
Discussion
Various phytochemical compounds which are naturally
present in plants as secondary metabolites have been
implicated in the conferment of antifungal activities
(Hostettmann & Marston, 1994; Grayer & Harborne,
1994; Osbourn, 1996; Al-Barwani & Eltayeb, 2004;
Athikomkulchal et al., 2006; Shanker et al., 2007, Fabri
et al., 2011). e presence of some of such secondary
metabolites in a signicant amount in the investigated
part of A. paniculata may have conferred the strong anti-
fungal activity on the whole plant extracts. In this regard,
higher concentration of these substances may have been
responsible for a higher degree of inhibition on the tested
strains. Previous studies have indicated that active prin-
ciples exhibit antimicrobial activity at a relatively lower
concentration in comparison to plant extracts; however,
plants which display antimicrobial activity at lower con-
centrations are considered potent antimicrobial plants
for further studies (Rios & Recio, 2005). Hence, the
inhibitory eects at low concentrations on the microor-
ganisms may in part be mediated through the chemical
constituents of the plant.
Results obtained from our study further conrm
the traditional use of A. paniculata against pathogenic
fungi. A. paniculata whole plant extracts displayed
prominent antifungal activity on the dermatophytes,
non-dermatophytes and yeasts strains tested. e
results also indicate that crude extract of the whole
plant A. paniculata exhibited prominent antifungal
activity. is behavior could be associated with a pos-
sible synergistic action of the compounds isolated from
this plant which is in accordance with previous studies
of antimicrobial activity of isolated compounds which
displayed stronger antimicrobial activity than the
plant extracts (Mbaveng et al., 2008; Sopa et al., 2008).
e use of dierent plant extracts in the treatment of
infections caused by various bacteria, viruses, and
fungi have already been reported and recognized
(Kosalec et al., 2005). Natural products, either as pure
compounds or as standardized plant extracts, provide
unlimited opportunities for new drug leads because of
the unmatched availability of chemical diversity (Cos
et al., 2006).
Conclusion
e TLC bioautography-guided strategy was eectively
used to separate the antifungal compounds from the
DCM and MEOH extracts of the whole plant of A. panic-
ulata. ree antifungal compounds were successfully
isolated. e isolated compounds, 3-O-β-d-glucosyl-
14-deoxyandrographiside, 14-deoxyandrographolide
and 14-deoxy-11,12-didehydrondrographolide, demon-
strated potent antifungal activities against the selected
microbial strains. Further investigation of the activities of
these compounds and their potential use in the treatment
of fungal diseases are still warranted. To our knowledge,
this is rst report on the isolation of antifungal substances
through bioassay-guided assay from A. paniculata.
Acknowledgement
Authors are grateful to the Research Management Center,
IIUM for nancial assistance through endowment grant
# EDW B 0904-267 to accomplish this work. Authors are
also grateful to Centre for Research and Instrumentation
Management, University Kebangsaan Malaysia (CRIM,
UKM), for conducting NMR analysis of all compounds.
Declaration of interest
e authors declare no conicts of interest.
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... Thus, there is paucity of information in respect to fungi contaminants on crop products and the use of plant to control fungi contaminants. Previous studies have used different solvents which include hexane, methanol, ethanol, etc. for extracting plant products [6,7,8]but studies on the use of local gin is scanty. The local gin is used for extraction of different herbs by locals. ...
... This agreed with the control in which the DMSO which served as negative control exerted no antifungal activity against the fungal isolates. Abubakaret al.[8] in their study demonstrated the antifungal activity of extracts of A. paniculata on Trichophyton mentagrophytes, Trichophyton rubrum, Microsporumcanis, Candida albicans, Candida krusei, Candida tropicalis, and Aspergillus niger which are skin pathogenic fungi. They associated the antifungal activity of A. paniculata to the synergistic actions of the compounds extracted from the plants. ...
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Testing and verifying the medicinal property of A. paniculata against fungal infestation
... A recent study also reported a similar number of unique secondary metabolites [169]. Among these compounds, about 80 compounds showed different types of pharmacological activities, including antibacterial [109,139,[170][171][172][173][174][175], anti-biofilm [88,176], antiviral (i.e., chikungunya virus [91], Anti-HIV [177][178][179], anti-influenza [99,180], Herpes simplex virus 1 [83,99,181], dengue virus serotype 1 [92][93][94]182], anti-Epstein Barr virus [97], pestivirus and flavivirus [90], human papillomavirus type 16 [96]), anti-fungal [109,183], antiparasitic (i.e., antimalarial [95,184,185], anti-Leishmaniasis [98]), antiproliferative [85][86][87]120,122,124,127,128,177,186], cytotoxicity [84][85][86]101,102,127,186], anti-inflammatory [84,86,136,187], antiplatelet aggregation [102], phagocytosis and anti-complement [84,86,102,104,109,113,114,117,128,133,135,138,142,177]. Some of these metabolites did not exhibit pharmacological properties or showed very week effectiveness. ...
... Sule, et al. [183] first reported on the isolation of antifungal bioactive compounds from dichloromethane (DCM) and methanol extracts of A. paniculata whole plant. All the isolated bioactive, 3-O-β-D-glucosyl-14-deoxyandrographolide, 14-deoxyandrographolide and 14deoxy-11,12-didehydroandrographolide, showed significant antifungal activity against Microsporum canis, A. niger, and C. albicans. ...
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Infectious disease (ID) is one of the top-most serious threats to human health globally, further aggravated by antimicrobial resistance and lack of novel immunization options. Andrographis paniculata (Burm. f.) Wall. ex Nees and its metabolites have been long used to treat IDs. Andrographolide, derived from A. paniculata, can inhibit invasive microbes virulence factors and regulate the host immunity. Controlled clinical trials revealed that A. paniculata treatment is safe and efficacious for acute respiratory tract infections like common cold and sinusitis. Hence, A. paniculata, mainly andrographolide, could be considered as an excellent candidate for antimicrobial drug development. Considering the importance, medicinal values, and significant role as antimicrobial agents, this study critically evaluated the antimicrobial therapeutic potency of A. paniculata and its metabolites, focusing on the mechanism of action in inhibiting invasive microbes and biofilm formation. A critical evaluation of the secondary metabolites with the aim of identifying pure compounds that possess antimicrobial functions has further added significant values to this study. Notwithstanding that A. paniculata is a promising source of antimicrobial agents and safe treatment for IDs, further empirical research is warranted.
... showed the MFC value at 50 μg/mL against M. canis. The DCM and methanolic extracts and their isolated compounds showed significant inhibitory effects (Sule et al., 2012). The crude extract of the leaves has been reported inhibition of the spore germination against Aspergillus flavus and Macrophomina phaseolina (Ghosh et al., 2004). ...
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... The plant showed potential therapeutic action in curing liver disorders, common cough, and colds in humans (nanduri et al., 2004;Akhtar et al., 2006;geethangili et al., 2008;Mishra et al., 2009;chandrasekaran et al., 2010;nagalekshmi et al., 2011;lim et al., 2012;sule et al., 2012). The present study was therefore performed to study the antioxidant and polyphenols of whole plant extract of A. paniculata in three different solvents, which may prove to be beneficial against free radical generated disorders. ...
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Problem statement: Chronic disease-causing bacteria of medical importance have developed resistance to antibiotics, hence, necessitating distinct and constant need for safe and efficient therapeutic agents. Plants are considered potent candidate for this aim. A way out of reducing antibiotic resistance and adverse effects on host is the employment of antibiotic resistance inhibitors of plant origin. Approach: About 5 kg pulverized Andrographis paniculata whole plant was macerated with MeOH at room temperature to get 305 g freeze dried MeOH extract. The bioautography of MeOH extract using Staphylococcus aureus and Proteus mirabilis as indicator organisms revealed the presence of two potent antibacterial compounds. MeOH extract was further fractionated and purified by silica gel column chromatography which led to the isolation of a diterpene lactone and an entlabdane diterpene glycoside upon crystallization with absolute ethanol. Results: Two antibacterial compounds viz., 3-O-β-D-glucosyl-14-deoxyandrographolide and 14-deoxyandrographolide were successfully isolated and characterized. Their structures were exclusively elucidated through spectroscopic methods (UV, IR, 1H- and 13C NMR). Conclusion: A. paniculata possesses antibacterial activity and could be potential source of a new class of antibiotics that might be useful for infectious disease chemotherapy and control.
Article
Herbs and spices have a traditional history of use, with strong roles in cultural heritage, and in the appreciation of food and its links to health. Demonstrating the benefits of foods by scientific means remains a challenge, particularly when compared with standards applied for assessing pharmaceutical agents. Pharmaceuticals are small-molecular-weight compounds consumed in a purified and concentrated form. Food is eaten in combinations, in relatively large, unmeasured quantities under highly socialised conditions. The real challenge lies not in proving whether foods, such as herbs and spices, have health benefits, but in defining what these benefits are and developing the methods to expose them by scientific means. Cultural aspects: The place of herbs and spices in the diet needs to be considered in reviewing health benefits. This includes definitions of the food category and the way in which benefits might be viewed, and therefore researched. Research may focus on identifying bioactive substances in herbs and spices, or on their properties as a whole food, and/or be set in the context of a dietary cuisine. The role of herbs and spices in health: The antioxidant properties of herbs and spices are of particular interest in view of the impact of oxidative modification of low-density lipoprotein cholesterol in the development of atherosclerosis. There is level III-3 evidence (National Health and Medical Research Council [NHMRC] levels of evidence1) that consuming a half to one clove of garlic (or equivalent) daily may have a cholesterol-lowering effect of up to 9%. There is level III-1 evidence that 7.2 g of aged garlic extract has been associated with anticlotting (in-vivo studies), as well as modest reductions in blood pressure (an approximate 5.5% decrease in systolic blood pressure). A range of bioactive compounds in herbs and spices have been studied for anticarcinogenic properties in animals, but the challenge lies in integrating this knowledge to ascertain whether any effects can be observed in humans, and within defined cuisines. Research on the effects of herbs and spices on mental health should distinguish between cognitive decline associated with ageing and the acute effects of psychological and cognitive function. There is level I and II evidence for the effect of some herbal supplements on psychological and cognitive function. There is very limited scientific evidence for the effects of herbs and spices on type 2 diabetes mellitus, with the best evidence being available for the effect of ginseng on glycaemia, albeit based on four studies. More research is required, particularly examining the effects of chronic consumption patterns. With increasing interest in alternatives to non-steroidal anti-inflammatory agents in the management of chronic inflammation, research is emerging on the use of food extracts. There is level II evidence for the use of ginger in ameliorating arthritic knee pain; however, the improvement is modest and the efficacy of ginger treatment is ranked below that of ibuprofen. More definitive research is required. Public health and dietary implications: Recommendations for intakes of food in the Australian guide to healthy eating2 do not yet include suggested intakes of herbs and spices. Future consideration should be given to including more explicit recommendations about their place in a healthy diet. In addition to delivering antioxidant and other properties, herbs and spices can be used in recipes to partially or wholly replace less desirable ingredients such as salt, sugar and added saturated fat in, for example, marinades and dressings, stir-fry dishes, casseroles, soups, curries and Mediterranean-style cooking. Vegetable dishes and vegetarian options may be more appetising when prepared with herbs and spices. Future directions: As several metabolic diseases and age-related degenerative disorders are closely associated with oxidative processes in the body, the use of herbs and spices as a source of antioxidants to combat oxidation warrants further attention. Immediate studies should focus on validating the antioxidant capacity of herbs and spices after harvest, as well as testing their effects on markers of oxidation. This will work in parallel with clinical trials that are aiming to establish antioxidants as mediators of disease prevention. From a dietary perspective, the functionality of herbs and spices will be exposed through consideration of their properties as foods. As with most foods, the real benefits of including them in the diet are likely to emerge with a better understanding of the attributes of health that are best supported by food, and in methodological developments addressing the evidence base for their effects. 3,4 These developments are well underway through evidence-based frameworks for substantiating health claims related to foods.5 At present, recommendations are warranted to support the consumption of foods rich in bioactive components, 6 such as herbs and spices. With Lime, we can expect to see a greater body of scientific evidence supporting the benefits of herbs and spices in the overall maintenance of health and protection from disease.
Article
Herbs and spices have a traditional history of use, with strong roles in cultural heritage, and in the appreciation of food and its links to health. Demonstrating the benefits of foods by scientific means remains a challenge, particularly when compared with standards applied for assessing pharmaceutical agents. Pharmaceuticals are small-molecular-weight compounds consumed in a purified and concentrated form. Food is eaten in combinations, in relatively large, unmeasured quantities under highly socialised conditions. The real challenge lies not in proving whether foods, such as herbs and spices, have health benefits, but in defining what these benefits are and developing the methods to expose them by scientific means. CULTURAL ASPECTS: The place of herbs and spices in the diet needs to be considered in reviewing health benefits. This includes definitions of the food category and the way in which benefits might be viewed, and therefore researched. Research may focus on identifying bioactive substances in herbs and spices, or on their properties as a whole food, and/or be set in the context of a dietary cuisine. THE ROLE OF HERBS AND SPICES IN HEALTH: The antioxidant properties of herbs and spices are of particular interest in view of the impact of oxidative modification of low-density lipoprotein cholesterol in the development of atherosclerosis. There is level III-3 evidence (National Health and Medical Research Council [NHMRC] levels of evidence) that consuming a half to one clove of garlic (or equivalent) daily may have a cholesterol-lowering effect of up to 9%. There is level III-1 evidence that 7.2 g of aged garlic extract has been associated with anticlotting (in-vivo studies), as well as modest reductions in blood pressure (an approximate 5.5% decrease in systolic blood pressure). A range of bioactive compounds in herbs and spices have been studied for anticarcinogenic properties in animals, but the challenge lies in integrating this knowledge to ascertain whether any effects can be observed in humans, and within defined cuisines. Research on the effects of herbs and spices on mental health should distinguish between cognitive decline associated with ageing and the acute effects of psychological and cognitive function. There is level I and II evidence for the effect of some herbal supplements on psychological and cognitive function. There is very limited scientific evidence for the effects of herbs and spices on type 2 diabetes mellitus, with the best evidence being available for the effect of ginseng on glycaemia, albeit based on four studies. More research is required, particularly examining the effects of chronic consumption patterns. With increasing interest in alternatives to non-steroidal anti-inflammatory agents in the management of chronic inflammation, research is emerging on the use of food extracts. There is level II evidence for the use of ginger in ameliorating arthritic knee pain; however, the improvement is modest and the efficacy of ginger treatment is ranked below that of ibuprofen. More definitive research is required. PUBLIC HEALTH AND DIETARY IMPLICATIONS: Recommendations for intakes of food in the Australian guide to healthy eating do not yet include suggested intakes of herbs and spices. Future consideration should be given to including more explicit recommendations about their place in a healthy diet. In addition to delivering antioxidant and other properties, herbs and spices can be used in recipes to partially or wholly replace less desirable ingredients such as salt, sugar and added saturated fat in, for example, marinades and dressings, stir-fry dishes, casseroles, soups, curries and Mediterranean-style cooking. Vegetable dishes and vegetarian options may be more appetising when prepared with herbs and spices. FUTURE DIRECTIONS: As several metabolic diseases and age-related degenerative disorders are closely associated with oxidative processes in the body, the use of herbs and spices as a source of antioxidants to combat oxidation warrants further attention. Immediate studies should focus on validating the antioxidant capacity of herbs and spices after harvest, as well as testing their effects on markers of oxidation. This will work in parallel with clinical trials that are aiming to establish antioxidants as mediators of disease prevention. From a dietary perspective, the functionality of herbs and spices will be exposed through consideration of their properties as foods. As with most foods, the real benefits of including them in the diet are likely to emerge with a better understanding of the attributes of health that are best supported by food, and in methodological developments addressing the evidence base for their effects. These developments are well underway through evidence-based frameworks for substantiating health claims related to foods. At present, recommendations are warranted to support the consumption of foods rich in bioactive components, such as herbs and spices. With time, we can expect to see a greater body of scientific evidence supporting the benefits of herbs and spices in the overall maintenance of health and protection from disease.