Bioassay-guided Isolation of Antifungal Plumericin from Allamanda
Farah F. Haron,
Anthracnose is an economically important disease that can
cause 10 to 80% yield reduction in the market with its typical symptoms of dark
spots, sunken necrotic tissues and concentric rings of acervuli on fruits. One
of the important methods to control the disease is by using antifungal agents
derived from plant. Preliminary study indicated that the extracts of Allamanda
species have the potential to be developed as the target agent. Five Allamanda
species were extracted and screened for antifungal activity against plant
pathogenic fungus Colletotrichum gloeosporioides by using poison agar
technique. The three chloroform extracts of A. blanchetti, A. cathartica
Alba and A. cathartica Jamaican Sunset exhibited
potent inhibitory effects and suppressed the mycelial growth of C. gloeosporioides
by up to about 70%. Further study by bioautography-guided fractionation of the
extracts led to the isolation and identification of tetracyclic sesquiterpene
plumericin as the antifungal agent. The structural determination of the compound
was carried out by interpreting the IR, MS, 1D-NMR and 2D-NMR spectral data.
July 27, 2012; Accepted: March 22, 2013;
Published: May 30, 2013
Genus Allamanda from Apocynaceae family is a popular warm climate plant
with trumpet-shaped flowers covering the whole plant throughout the year, easily
cultivated, fast growing and gives beauty to any landscape. It is woody evergreen
tree with vigorous growth. Allamandas are different in shapes and sizes
from small shrubs, big shrubs to vines that can cover the whole fence with beautiful
flowers when bloom. The genus is native to South and Central America and had
become neutralized throughout the tropical areas (Min et
al., 2006). In traditional herbal medicine, leaves and roots extracts
of Allamanda cathartica are used as a strong purgative, treating malaria,
jaundice and enlarged spleen (Liogier, 1995; Nayak
et al., 2006). The extracts were also reported to possess remarkable
wound healing property for treating various types of wounds in human besides
its antibiotic action against Staphylococcus (Nayak
et al., 2006). Allamanda species has been demonstrated as
antifungal, antileukemic, anti-HIV (Kardono et al.,
1990; Tan et al., 1991), anticancer (Dobhal
et al., 2004), cytotoxic activity against Madison lung carcinoma
(Abdel-Kader et al., 1997) and strong fungitoxicity
against some dermatophytes causing dermatomycosis in animals and humans (Tiwari
et al., 2002).
The major postharvest disease of tropical fruits is anthracnose caused by Colletotrichum
gloeosporioides and the infections can occur on immature and ripe fruits.
To reduce this problem, fungicides were applied either at preharvest or postharvest
period (Dodd et al., 1989; Muirhead
and Gratitude, 1986). However, the damage it causes is more important in
the postharvest period. Due to public concerned about toxic effect of fungicides,
their uses had been restricted. Moreover, few farmers in developing countries
can afford to purchase these expensive chemicals. Biocontrol agents are economically
cheaper and suitable for small scale farmers in developing countries and offer
an attractive alternative to overcome this problem. Only a few biocontrol agents
have been reported for postharvest protection of tropical fruits against diseases
(Govender and Korsten, 2006; Jager
et al., 2001; Koomen and Jeffries, 1993).
The primary move in finding new biocontrol agents and agrochemicals is bioassay-directed
screening of plant extracts and compounds obtained from them. Since, there has
been no report on the major active antifungal substances in Allamanda
species extracts responsible for controlling C. gloeosporioides, causal
agent of papaya anthracnose, the aim of the present study is to evaluate the
antifungal activity of Allamanda species and bioassay-guided separation
of the active components.
MATERIALS AND METHODS
Instrumentation: This research work was conducted between 2009 and 2012.
The IR spectra were recorded on a Perkin Elmer FTIR 1650 spectrophotometer using
KBr discs measured in cm-1; MS were recorded on a SHIMADZU GC-MS
QP2010 Plus with EI electron impact ion source of 70eV; 1H-NMR 13C-NMR,
DEPT-135, HMQC, HMBC, COSY NMR spectra were obtained on a Varian AS 400 spectrometer
using CDCl3 as solvent; High Performance Flash Chromatography (HPFC)
was performed using a Biotage Inc. Horizon pump (Charlottesville, VA) equipped
with a Horizon flash collector and a fixed wavelength (254 nm) detector (system
from Biotage Inc. Dyax Corp.). Biotage columns (surface area 500 m2
g-1, porosity 60 Å, particle size 40-63 μm) used were
made up of FLASH 12+M, KP-SiL (12x150 mm) and FLASH 25+M, KP-SiL (25x150 mm);
TLC was carried out on commercial TLC plates with fluorescent indicator (250
μm, silica gel GF Uniplate, Analtech Inc., Newark DE), developing with
solvent mixture made up of EtOAc and hexane, visualization either under UV light
at 254 or 365 nm and/or by spraying with anisaldehyde and heating at 110°C.
Statistical analysis: The treatments were arranged in a completely randomized
design in four replicates with Allamanda spp., types of extracts and
concentration levels as factors. All data were subjected to analysis of variance
(ANOVA) where significant (p<0.05) differences between means were determined
by Tukeys standardized range
test (HSD). Relationships (quadratic) between colony diameter and extract concentration
were determined by regression analysis of data obtained for the different Allamanda
species and types of extracts. The SAS (version 9.2) software was used to perform
Plant materials: Allamanda blanchetti, Allamanda cathartica,
Allamanda cathartica Alba,
Allamanda cathartica Jamaican
Sunset and Allamanda oenotheraefolia
were obtained from nurseries in Sungai Buloh, Selangor and University Agriculture
Park, UPM Serdang, Malaysia. The leaves were washed, air-dried at room temperature,
ground into powder and stored until required for extraction.
Plant extraction and bioassay: The dried fine powdered leaf of each
plant (1 kg) was separately and sequentially extracted with petroleum ether,
chloroform and methanol at room temperature. The extracts were filtered through
Whatman no. 1 filter paper and concentrated by rotary evaporator to give fifteen
dark sticky semisolid extracts. The stock solutions (100 mg mL-1)
were serially diluted with acetone to obtain the desired concentration for test
solutions of 1, 3, 5 and 7 mg mL-1. Effect of Allamanda extracts
on mycelia growth of C. gloeosporioides was measured in vitro
according to the method described by Bautista-Banos et
al. (2003) using poison agar technique. PDA was amended with 4 concentrations
of each extract (1, 3, 5 or 7 mg mL-1). Fifteen mL of the amended
PDA was poured into petri dishes (9 cm) and a fungal plug (4 mm) from pure culture
of C. gloeosporioides was placed in the centre of the dishes. Petri dishes
were then incubated at room temperature and radial measurements of growth were
recorded daily until the fungus reached the edge of the plate. Petri dishes
containing PDA and acetone served as control. Percentage Inhibition of Radial
Growth (PIRG) was calculated after 8 days of incubation using a standard formula:
where, R1 is the radial growth of fungus in the control plate (mm),
while R2 is the radial growth of the fungus containing extracts (mm)
(Sivakumar et al., 2000).
Bioautography-guided fractionation and isolation of plumericin: The
chloroform extract of A. cathartica Jamaican
Sunset (5 g) was absorbed into
silica gel Biotage samplet and applied to a Biotage column FLASH
25+M, KP-SiL (150x25 mm i.d.; 40 mL min-1). Elution of the column
was performed by using mixtures of EtOAc:hexane (1:9, 3:7; 1:1; 7:3; 9:1) to
give 240 fractions of 50 mL each. Based on TLC analysis, similar fractions were
combined and their activity was monitored by bioautography. The active combined
fractions 86-112 were further separated by preparative TLC (20x20 cm) and eluted
twice with ether:hexane (1:1) and the two active bands were scraped off separately.
The scraped bands were dissolved in dichloromethane, filtered through cotton
wool to give two white solids. Due to insufficient amount of one of the samples,
only the major and active component was submitted for further spectroscopic
analysis and identified as plumericin. IR vmax cm-1 (CHCl3):
2921, 2854 (CH), 1759 (C = O), 1696 (C = O), 1448 (C = C); 1H-NMR
(CDCl3, 400 MHZ)-see Table 1; 13C-NMR
(CDCl3, 400 MHZ)-see Table 1; EIMS m/z (%): 290
(M+) (35), 272 (30), 258 (60), 230 (100), 212 (20), 193 (60), 173
(45), 160 (70), 145 (60), 139 (85), 115 (55), 105 (40).
||1H-NMR (400 MHZ, CDCl3) and 13C-NMR
(100 MHZ, CDCl3) spectral data of plumericin
|*Brigitta et al. (2005)
Bioautography: TLC plates for bioautography assays were spotted with
80 μg of crude plant extracts and selected fractions and then chromatographed
in duplicate (Fig. 1). Fungicide technical grade standards
benomyl, cyprodinil, captan (Chem Service, Inc., West Chester, PA) and azoxystrobin
(Syngentia, Greensboro, NC) were used as controls at 2 μg in 4 μL
of acetone. TLC plates were sprayed with spore suspension adjusted to a final
concentration of 1.0x06 conidia mL-1 with liquid potato
dextrose broth (PDB, Difco) and 0.1% Tween-80 in order to detect biological
activity directly on the TLC plate. Using a 50 mL chromatographic sprayer, each
TLC plate with a fluorescent indicator was sprayed lightly with the conidial
suspension. Inoculated plates were then placed in a 30x13x7.5 cm moisture chamber
(25°C, 100% relative humidity, Pioneer Plastics, Inc., Dixon, KY) and incubated
in a growth chamber at 24±1°C. Inhibition of fungal growth was measured
4 days after treatment. The sensitivity of the fungus to each test sample was
indicated by the presence of clear zones of fungal growth inhibition directly
on TLC plates.
RESULTS AND DISCUSSION
The petroleum ether, chloroform and methanol extracts of the five plants were
monitored for antifungal activity by poison agar technique and the three chloroform
extracts of A. blanchetti (CEB), A. cathartica Alba
(CEA) and A. cathartica Jamaican Sunset (CEJS) with strong
antifungal activity were selected for further investigation. TLC development
of these extracts eluted with ethyl acetate:hexane (3:7) revealed the presence
of several prominent spots in each plate with the same Rf values
range from 0.17-0.77 (Fig. 1a). The plates were then sprayed
with C. gloeosporioides and incubated for 4 days at 24±1°C.
|| (a) TLC chromatograms and (b) Bioautograms of A. cathartica
Alba (CEA), A. blanchetti (CEB) and A. cathartica
Jamaican Sunset (CEJS) chloroform extracts against C. gloeosporioides
in 30% ethyl acetate:hexane as solvent system. (Clear zones indicate inhibition
of fungal growth by compounds in the extracts after 4 days of incubation
Very clear and prominent zones were visible on three of the active spots with
the same Rf values (0.3-0.55) on the bioautograms (Fig.
1b). This clearly indicated the presence of phytochemicals which inhibited
the growth of C. gloeosporioides. The chloroform extract of A. cathartica
Jamaican Sunset (CEJS) was selected for larger scale separation
by using High Performance Flash Chromatography (HPFC) with Biotage column
FLASH 12+M, KP-SiL (12x150 mm) and eluted with various mixtures of solvents
to give 240 fractions of 50 mL each. All fractions collected were monitored
by bioautography and the active fractions (86-112) with prominent clear zones
were combined and further purified by preparative TLC.
|| Structure of plumericin
The major and prominent active spot was scraped, dissolved in dichloromethane,
filtered and on solvent removal white solid was obtained. Since the Rf
values of the active spots for the two other chloroform extracts of A.
blanchetti and A. cathartica Alba were identical to the
isolated compound, no further separation work was carried on them.
Confirmation of the structure of the isolated compound (Fig.
2) was carried out by comparing the spectral data obtained with literature
values. The IR spectrum revealed existence of two carbonyl groups as indicated
by the occurrence of prominent absorptions at 1696 and 1759 cm-1.
The mass spectrum showed a molecular ion peak at m/z 290 which corresponded
to the molecular formula C15H14O6 and the base
peak occurred at m/z 230 due the cleavage of -CO2CH3.
The integration of 1H-NMR indicated the presence of fourteen protons
which further supported the suggested molecular formula (Table
1). The two methyl groups observed at δ 2.08 (day) and 3.77 (sec) are
assigned to C-14 and C-16, respectively. The rest of the proton signals are
due to the occurrence of eight methine groups and they are assigned based on
coupling constants, COSY and HMBC correlations spectra. Two of them are observed
as singlets at δ 7.44 (H-3) and 5.10 (H-10). Another two occurred as doublets
at δ 5.56 and 6.04 assigned for H-1 and H-7. A quartet observed at δ
7.16 which exhibited 2J correlation with the carbonyl group (C-12)
is assigned to H-13. The 2J and 3J correlations between
protons and carbons observed in the HMBC spectrum further supported the assignments
of other methine groups. The attachment of the carboxylate group to the pyran
ring is rationalized by the correlation of H-3 to the carboxyl carbon. The existence
of fifteen carbon atoms displayed by the 13C-NMR and DEPT spectra
are made up of two methyls, eight methines, three quarternary carbons and two
carbonyl groups. The assignments of various protons to their respective carbon
atoms could be clearly seen in the HMQC spectrum. Based on these detailed spectral
analysis and comparison with literature report, the compound is identified plumericin
previously isolated and identified from Plumeria rubra (Brigitta
et al., 2005).
Plumericin is an important member of tetracyclic sesequiterpene commonly found
in various plant families including from different genera of Apocynaceae such
as Plumeria, Allamanda and Himatanthus (Silva et al.,
2010; Sharma et al., 2011). The antifungal,
anticancerous, antiviral and antibacterial actions of plumericin have been reported
previously (Singh et al., 2011; Sticher,
1977). The compound isolated from another species within the Apocynaceae
(Himatanthus sucuuba) has been demonstrated to have antiparasitic activity
against Leishmanial amazonnensis, responsible for cutaneous leishmaniasis
(Castillo et al., 2007). It is believed that
the strong activity of the tetracyclic plumericin is due the presence of an
á-methylene-γ-lactone moiety which is susceptible to undergo a Michael-type
addition with biological nucleophiles (Kupchan et al.,
1970). Knobloch et al. (1986) reported that
sesquiterpenes affect the bacterial processes that include the inhibition of
electron transport, protein translocation, phosphorylation steps and other enzyme-dependent
reactions. Most probably similar mechanisms of action were responsible for the
antifungal activity of the compound under study. Structural characteristics
and chemical properties of terpenes and fatty acids played major roles in signifying
antifungal activity for compositions containing these compounds with the presence
of oxygenated substituent such hydroxyl and carboxyl groups (Glinski
and Branly, 2002).
Antifungal-guided isolation of the chloroform extract of A. cathartica
Jamaican Sunset has led
to the identification of plumericin as the compound responsible for the potent
antifungal activity exhibited by the plant. The structure of the compound was
determined by detailed spectroscopic analysis and comparison with previous reports
and has the potential to control anthracnose disease caused by pathogenic fungus
Colletotrichum gloeosporioides, an economically important disease in
postharvest papaya cultivation. This study adds another important development
of this interesting compound which has been the target of various chemical syntheses.
We gratefully acknowledge Dr. S.O. Duke, Dr. K.M. Meepagala, Mr. J. Martin
and Ms. L. Robertson from USDA-ARS-NPURU for technical assistance. We also would
like to acknowledge Universiti Putra Malaysia for providing facilities to carry
out this project.
Abdel-Kader, M.S., J. Wisse, R. Evans, H. van der Werff and D.G.I. Kingston, 1997. Bioactive iridoids and new lignan from Allamanda cathartica and Himatanthus fallax from the Suriname rainforest. J. Nat. Prod., 60: 1294-1297.
CrossRef | PubMed |
Bautista-Banos, S., M. Hernandez-Lopez, E. Bosquez-Molina and C.L. Wilson, 2003. Effects of chitosan and plant extracts on growth of Colletotrichum gloeosporioides, anthracnose levels and quality of papaya fruit. Crop Protect., 22: 1087-1092.
CrossRef | Direct Link |
Castillo, D., J. Arevalo, F. Herrera, C. Ruiz and R. Rojas et al., 2007. Spirolactone iridoids might be responsible for the antileishmanial activity of a Peruvian traditional remedy made with Himatanthus sucuuba (Apocynaceae). J. Ethnopharmacol., 112: 410-414.
CrossRef | Direct Link |
Dobhal, M.P., G. Li, A. Gryshuk, A. Graham and A.K. Bhatanager et al., 2004. Structural modifications of plumieride isolated from Plumeria bicolor and the effect of these modifications on In vitro anticancer activity. J. Org. Chem., 69: 6165-6172.
CrossRef | PubMed |
Dodd, J.C., P. Jeffries and M.J. Jeger, 1989. Management strategies to control latent infectionin tropical fruits. Asp. Applied Biol., 20: 49-56.
Direct Link |
Elsasser, B., K. Krohn, M.N. Akhtar, U. Florke and S.F. Kouam et al., 2005. Revision of the absolute configuration of plumericin and isoplumericin from Plumeria rubra. Chem. Biodiver., 2: 799-808.
CrossRef | Direct Link |
Glinski, J. and K.L. Branly, 2002. Pentacyclic triterpenes. U.S. Patent No. 6303589. http://www.patentstorm.us/patents/6303589.html.
Govender, V. and L. Korsten, 2006. Evaluation of different formulations of Bacillus licheniformis in mango pack house trials. Biological Control, 37: 237-242.
CrossRef | Direct Link |
Jager, E.S., F.C. Wehner and L. Karsten, 2001. Microbial ecology of the mango phylloplane. Microbial. Ecol., 42: 201-207.
Kardono, L.B., S. Tsauri, K. Padmawinata, J.M. Pezzuto and A.D. Kinghorn, 1990. Cytotoxic constituents of the bark of Plumeria rubra collected in Indonesia. J. Nat. Prod., 53: 1447-1455.
PubMed | Direct Link |
Knobloch, K., N. Weigand, H.M. Weis and H. Vigenschow, 1986. Progress in Essential Oil Research. Walter de Gruyther, Berlin, Germany.
Koomen, I. and P. Jeffries, 1993. Effects of antagonistic micro organisms on the post harvest development of Colletotrichum gloeosporioides on mango. Plant Pathol., 42: 230-237.
Kupchan, S.M., T.J. Giacobbe, J.S. Krull, A.M. Thomas, M.A. Eakin and D.C. Fessler, 1970. Reaction of endocyclic α, β- unsaturated lactones with thiols. J. Org. Chem., 35: 3539-3543.
Liogier, H.A., 1995. Descriptive Flora of Puerto Rico and Adjacent Islands. de la Universidad de Puerto Rico, San Juan, Puerto Rico.
Min, B.C., K. Omar-Hor and O.Y.C. Lin, 2006. 1001 Garden Plants in Singapore. 2nd Edn., National Park Board, Singapore.
Muirhead, I.F. and R. Gratitude, 1986. Mango diseases. Proceedings of the 1st Australian Mango Research Workshop, November 26-30, 1986, Cairns, Australia, pp: 248-252.
Nayak, S., P. Nalabothu, S. Sandiford, V. Bhogadi and A. Adogwa, 2006. Evaluation of wound healing activity of Allamanda cathartica L. and Laurus nobilis L. extracts on rats. BMC Complement. Altern. Med., Vol. 6. 10.1186/1472-6882-6-12
Sharma, U., D. Singh, P. Kumar, M.P. Dobhal and S. Singh, 2011. Antiparasitic activity of plumericin and isoplumericin isolated from Plumeria bicolor against Leishmania donovani. Indian J. Med. Res., 134: 709-716.
Silva, J.R.A., C.M. Rezende, A.C. Pinto and A.C.F. Amaral, 2010. Cytotoxicity and antibacterial studies of iridoids and phenolic compounds isolated from the latex of Himatanthus sucuuba. Afr. J. Biotechnol., 9: 7357-7360.
Direct Link |
Singh, D., U. Sharma, P. Kumar, Y.K. Gupta, M.P. Dobhal and S. Singh, 2011. Antifungal activity of plumericin and isoplumericin. Nat. Prod. Commun., 6: 1567-1568.
Sivakumar, D., R.S.W. Wijeratnam, R.L.C. Wijesundera, F.M.T. Marikar and M. Abeyesekere, 2000. Antagonistic effect of Trichoderma harzianum on postharvest pathogen of rambutan (Nephelium lappaceum). Phytoparasitica, 28: 240-247.
Sticher, O., 1977. Plant Mono, Di and Sesquiterpenoids with Pharmacological or Therapeutical Activity. In: New Natural Products with Pharmacological, Biological or Therapeutical Activity, Wagner, H. and P. Wolff (Eds.). Springer Verlang, Berlin, Germany, pp: 137-176.
Tan, G.T., J.M. Pezzuto, A.D. Kinghorn and S.H. Hughes, 1991. Evaluation of natural products as inhibitors of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase. J. Nat. Prod., 54: 143-154.
PubMed | Direct Link |
Tiwari, T.N., V.B. Pandey and N.K. Dubey, 2002. Plumieride from Allamanda cathartica as an antidermatophytic agent. Phytother. Res., 16: 393-394.
CrossRef | PubMed |