International Journal of Pharmacy and Pharmaceutical Sciences

ISSN- 0975-1491 Vol 8, Issue 10, 2016

Original Article
ANTIFUNGAL ACTIVITY OF PIMENTA DIOICA (L.) MERRIL AN AROMATIC MEDICINAL TREE

AKHILESH BHAT, RAVEESHA K. A.*

Centre for Innovative Studies in Herbal Drug Technology, Department of Studies in Botany, University of Mysore, Manasagangotri,
Mysore 570026
Email: [email protected]
Received: 17 Nov 2015 Revised and Accepted: 12 Aug 2016
ABSTRACT
Objective: To assess the antifungal activities of the extracts of leaves of Pimenta dioica against human pathogenic fungi and identify the class of
phytochemical responsible for the biological activity.
Methods: The shade dried, and powdered leaves of Pimenta dioica were subjected to polarity based solvent extraction by soaking. The extracts
were dried in a rotary flash evaporator and subjected to antifungal activity assay against Candida albicans, Microsporum canis and M. gypseum, by
disc diffusion method. Antifungal active petroleum ether extract was subjected to phytochemical analysis to identify the active compound. The
extract was then subjected to Thin Layer Chromatography (TLC) and Bioautography, by agar overlay method. The fractions were collected by
Preparative TLC method and subjected to antifungal activity assay by disc diffusion method. Further, the Libermann-Burchard test was conducted
to detect the broad group of compounds responsible for the bioactivity.
Results: Among the five extracts viz. petroleum ether, chloroform, ethyl acetate, methanol and aqueous, the petroleum ether extract showed
significant antifungal activity against Microsporum canis (50 mm), Microsporum gypseum (60 mm) and Candida albicans (23 mm). Terpenoids,
tannins and flavonoids were found to be the chief constituents of the petroleum ether extract. The TLC of the extract revealed 7 spots with the Rf
values 0.37, 0.42, 0.45, 0.52, 0.59, 0.76, 0.96 with the solvent system of hexane and acetone (1:0.5). The bioautography assay revealed inhibitory
activity in the spots at Rf values 0.52 and 0.59. These fractions obtained by preparative TLC also confirmed the same. The phytochemical analysis of
the fractions on the TLC plate suggests that the compound is triterpene or steroid.
Conclusion: The antifungal activity of Pimenta dioica and the bioactive phytochemical has been reported in the present study. Preliminary
phytochemical analysis has indicated that the compounds are triterpenes or steroids. The results indicate that the active compounds are potential
lead molecules for new antifungal drugs in the management of infectious fungi.
Keywords: Pimenta dioica, Leaf extract, Terpenoids, Bioautogrpahy, Antifungal activity, Microsporum, Candida
© 2016 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons. org/licenses/by/4. 0/)
DOI: http://dx.doi.org/10.22159/ijpps.2016v8i10.9924

INTRODUCTION In this context, plants can be exploited for discovering biomolecules

with therapeutic potential against human disease-causing fungi. The
History is replete with references to human infectious diseases, plants generally used in traditional health care are known to possess
which have altered its course. At the same time, man has also been in a wide array of bioactive molecules with low or limited side effects.
a constant quest for effective medicines against these diseases. It may also be recalled that plants are the chief source of
Preparations of medicinal plant parts were some of the earliest biomolecules for therapeutic drugs; as much as 40% of all known
therapeutics employed. Many of these preparations were preserved drugs are either directly sourced from plants or the derivatives of
in writing and are available to this day. plant biomolecules [4].
However, some of the medicinal uses of plants are not recorded in The aim of the study was to analyze the antifungal potential of the
literature, but the knowledge has been passed through generations extracts of the leaves of the aromatic tree Pimenta dioica (L.) Merril.
of healers in the various indigenous communities. All these It is an exotic plant whose fruits are used as a spice and its aroma
knowledge sources are an important resource in the development of and flavour is a mix of Clove, Cinnamon and Nutmeg. Hence it is
new drugs to combat infectious diseases. commonly referred as “allspice”.
Among the various infectious diseases afflicting humans, the Allspice or Pimenta dioica is small-sized tree belonging to the family
diseases caused by fungi are very difficult to cure and require a Myrtaceae. It is a small tree, about 8-10 m tall with smooth, peeling
prolonged duration of treatment. They are a significant cause of bark. The leaves are oppositely arranged, petiolate, with narrowly
morbidity and mortality in immunocompromised patients who elliptic or oblong blades having abundant oil glands and strong,
includes patients with AIDS, cancer and those with organ transplants spicy odor when crushed. They are dioecious; flowers are borne in
[1]. Fungal diseases in humans can be either subcutaneous or axillary panicles; sepals are distinct in bud; petals and stamens are
systemic. The present study focuses on fungi, which are primarily white colored. The fresh fruits are green, with purple-black colour
subcutaneous. on ripening; two seeds are found in each fruit [5].

The existing treatment for fungal diseases comprises of a class of The essential oils of the leaves and fruits of this plant are anesthetic,
steroidal drugs named “Azole drugs”. Therapeutic options for analgesic, antimicrobial, antioxidant, antiseptic, acaricidal,
treatment are limited, and most of these drugs have similar modes of carminative, muscle relaxant, rubefacient, and stimulant. It is also
action. Further, these steroidal drugs are known to possess a used as a tonic for several digestive tract problems like cramps,
number of side effects and present problems of toxicity over flatulence, indigestion and nausea. Further, its essential oils are used
prolonged treatment durations. Furthermore, most treatments are in medicines to treat cases of depression, nervous exhaustion,
not capable of completely eradicating the fungus from the infected tension, neuralgia and stress. Commercially, the leaf and fruit oil is
individual’s body [2, 3]. Discovering new antifungal agents with used in perfumery, aftershaves and food flavorings [6-8]. Since the
potent activity and low toxicity is the need of the hour. plant is widely used in traditional medicine as antimicrobial and
 Raveesha et al.
Int J Pharm Pharm Sci, Vol 8, Issue 10, 92-95

antiseptic, it has been selected for scientific validation in the present Phytochemical analysis
study.
The preliminary phytochemical analysis of the petroleum ether
MATERIALS AND METHODS extract of the leaves of P. dioica was done to identify the broad
groups of phytochemicals present in the extract. The tests were
Test plant
carried out according to the procedures outlined by Trease and
The leaves of P. dioica were collected from a fully grown tree in a Evans, Sofowora, and Harborne [12-14].
farm at Maratikyathanahalli, Mysore. It was identified by an expert
Thin layer chromatography
taxonomist of Department of Studies in Botany, and a herbarium
specimen is deposited in the herbarium of the Department of Studies The petroleum ether extract of the leaves of P. dioica, which
in Botany, University of Mysore. The leaves were washed with showed significant antifungal activity, was subjected to TLC to
running tap water; shade dried, powdered and sealed and stored at separate the phytochemicals in it. Various combinations of the
4˚C until used. polar and non-polar solvents were tried to determine the best
solvent system to achieve a clear separation of the bands. The
Preparation of plant extracts Retention factor (Rf) values of the separated bands was calculated
The dried leaf powder was extracted successively with increasingly by using the formula:
polar solvents viz. petroleum ether, chloroform, ethyl acetate,
methanol (Thermo Fischer Scientific, Mumbai) and water. 100 g of
the leaf powder was soaked in 200 ml of the solvent for 24 h and
filtered through Whatman No.1 filter paper (Whatman International Bioautography
Ltd., Maidstone, England). The filtrate was dried in a rotary flash
evaporator (IKA-RV10) and the dried extract was stored at 4 °C until The agar overlay method was used to conduct the bioautography
further use [9]. assay to localize the antifungal compound on the TLC plate after
separation. TLC was carried out on silica gel-250-GF pre-coated
Test fungi aluminium plates (Merck KGaA, Darmstadt, Germany) of 7 cm x 2 cm
Candia albicans (yeast) (MTCC183), Microsporum canis (MTCC2820) dimensions. The plates were kept under UV light for sterilization and
and Microsporum gypseum (MTCC2830) (dermatophytes) were then placed on the lower lid of sterile petriplates. SDB with 0.7%
selected as test organisms for the present study. The test fungi were w/v agar medium inoculated with the test fungi was poured
procured from Microbial Type Culture Collection Centre (MTCC), carefully on the TLC plates. The plates were sealed and incubated at
Institute of Microbial Technology, Chandigarh the organisms were 35±2 ˚C for 48 h [3].
maintained on Sabourad’s Dextrose Agar (SDA) (HiMedia, Mumbai).
Phytochemical analysis of the TLC spots 0.52 and 0.59
Antifungal activity assay
After separation of the compounds in the petroleum ether extract by
The antifungal activity assay of aqueous extracts was done by agar- TLC, the TLC plates were sprayed with modified Liebermann–
well diffusion method [10] and that of the solvent extracts was done Burchard reagent (1 ml conc. Sulphuric acid+20 ml Acetic
by disc diffusion method [11]. Twenty-four hours old culture of C. anhydride+25 ml Chloroform) and placed in an oven at 85-90 ˚C for
albicans and ten-day old cultures of dermatophytes grown in 15 min [14].
Sabourad’s Dextrose Broth (SDB) were taken as inoculum for the
RESULTS
assays. Miconazole (HiMedia, Mumbai) was employed as a positive
control. The concentration of the solvent extracts and the positive Antifungal activity assay
control was 10 mg/ml and 50 µl was added to each disc. 100 µl of
the aqueous extracts was added to the agar wells. Negative control The antifungal activity assay (table 1) indicated a strong antifungal
was maintained by taking the respective solvents used for the effect in the petroleum ether extract of the leaves of P. dioica. The
preparation of the extracts. Three replicates were maintained. diameters of the zone of inhibition against the test fungi were 23
mm, 50 mm and 60 mm against C. albicans, M. canis and M. gypseum
Statistical analysis respectively. The activity was comparable to that of the positive
control Miconazole.
The results of the antifungal activity assays were statistically
analyzed by using the software SPSS (Software Package for Social The chloroform extract also had some inhibitory effect on all the
Sciences) ver.20 (IBM Corp., Armonk, New York). The data was test fungi with zone sizes ranging from 20 mm to 40 mm. The
subjected to One-Way ANOVA (Analysis of Variance) and Tukey’s methanol extract showed mild inhibition of the growth of the
HSD (Highest Significant Difference) at P<0.05. dermatophytes.

Table 1: Antifungal activity of the extracts of leaves of Pimenta dioica against human pathogenic fungi
Extract Diameter of the zone of Inhibition (mm)
Candida albicans Microsporum canis Microsporum gypseum
Petroleum ether 23.3±0.33c 50.3±0.33e 59.6±0.33d
Chloroform 19.3±0.33b 19.6±0.33c 39.6±0.33c
Ethyl Acetate 0±0.0a 0±0.0a 0±0.0a
Methanol 0±0.0a 13.3±0.33b 10.3±0.33b
Water 0±0.0a 0±0.0a 0±0.0a
Miconazole 50 µg, (Positive control) 23.6±0.33c 39.6±0.33d 40.3±0.33c
Note: Concentration of the test extracts is 0.1 g/ml, All values are mean±SE of triplicates. The values with different superscripts are significantly
different from one another at P<0.05 (Tukey’s HSD).

The statistical analysis of the results of the antifungal activity assay Phytochemical analysis
revealed that the activity of petroleum ether extract was
The results of the phytochemical analysis are presented in table 2.
significantly higher than that of the positive control (miconazole) The broad groups of phytochemicals present in the petroleum ether
against M. canis and M. gypseum and comparable with it in the case extract of the leaves of P. dioica are terpenoids, steroids, coumarins,
of C. albicans. tannins and flavonoids.

93
 Raveesha et al.
Int J Pharm Pharm Sci, Vol 8, Issue 10, 92-95

Table 2: The phytochemical analysis of the petroleum ether

extract of the leaves of P. dioica
Phytochemical Presence/absence
Carbohydrates --
Proteins and amino acids --
Phenols --
Tannins +
Saponins --
Phlobatannins --
Alkaloids --
Flavonoids +
Terpenoids +
Steroids +
Cardiac Glycosides --
Glycosides --
Carotenoids --
Coumarins +
Anthraquinones --
Fig. 3: The Phytochemical analysis of the TLC spots 0.52 and
Note: “+” indicates presence, “--” indicates absence
0.59. A. TLC of the Petroleum ether extract, B. Plate sprayed
with reagent showing violet spots at Rf 0.52 and 0.59

Thin layer chromatography

The petroleum ether extract of P. dioica showed the presence of Bioautography
seven bands when subjected to TLC with the solvent system The bioautography of the petroleum ether extract of P. dioica
comprising of hexane and acetone in the ratio 1: 0.5. The Rf values of showed small clear zones around the TLC spots at Rf 0.52 and 0.59
the bands were calculated, and they were as follows: 0.37, 0.42, 0.45, (fig. 2) against all the three test fungi. It indicated that the antifungal
0.52, 0.59, 0.76, 0.96 (fig. 1). compounds are present at the above-mentioned positions on the
TLC plates. However, further separation would be needed to
characterize the compounds and assess their potency in inhibiting
the growth of the test fungi.
Phytochemical analysis of the TLC spots 0.52 and 0.59
The TLC plates sprayed with modified Liebermann–Burchard
reagent showed a change in the colour of the spots from dark green
to bluish-green indicating the presence of triterpenes/steroids.
DISCUSSION
The study analyses the potential of the extracts of the leaves of P.
dioica in inhibiting the growth of selected human pathogenic fungi;
the phytochemical analysis of the petroleum ether extract which
showed the highest antifungal activity and separation and
localization of the bioactive compounds by TLC and bioautography.
Fig. 1: Thin layer chromatography of the petroleum ether
extract of P. dioica. A. under visible light, B. after treatment with Reports on the pharmacological activities, particularly antimicrobial,
iodine vapour, C. under long UV light (365 nm), D. under short antioxidant and tumoricidal activity of different parts of the plant
UV light (254 nm) are available in the literature, and a few bioactive compounds have
been isolated and characterized. Al-Rehaily et al. [15] conducted
studies on the pharmacological properties of the aqueous
suspension of fruits of P. dioica for anti-inflammatory, analgesic,
antipyretic, gastric antiulcer, and cytoprotective activities in
experimental models. Their studies showed that the extract had
anti-inflammatory, cytoprotective and antiulcer activities. More
importantly, toxicity studies showed neither mortality nor adverse
effects up to a dose of 7.5 g/kg in mice. Thus the study established
that P. dioica fruit extracts may be less toxic and effective
therapeutically.
Lai and Roy [16] reviewed the existing literature of that time on the
antimicrobial and chemopreventive properties of some commonly
used herbs and spices, including P. dioica. They have collected data
of reported activities against mycotoxigenic Aspergillus, Shigella
sonnei and Shigella flexneri, Salmonella entiritica and Listeria
monocytogens. Eugenol was found to be the most widely attributed
active compound from these studies.
The chemical composition of the essential oil extracted from the
leaves of P. dioica from Jamaica was analyzed by GC and GC-MS [8].
Eugenol (76.02%), methyl eugenol (7.14%) and β-caryophyllene
Fig. 2: Bioautography of the petroleum ether extract of P. dioica (6.47%) were found to be the major constituents.
against test fungi. A. Microsporum canis, B. Microsporum
gypseum, C. Candida albicans, D. TLC plate with the active spots The antimicrobial activity of essential oil of P. dioica and other spices was
marked evaluated against spoilage and pathogenic microorganisms isolated from

94
 Raveesha et al.
Int J Pharm Pharm Sci, Vol 8, Issue 10, 92-95

meat products, which included many bacteria such as Staphylococcus 2. Sheehan DJ, Hoelscher AC, Sibley MC. Current and emerging
aureus, Pseudomonas aeruginosa, Escherichia coli, etc., along with the azole antifungal agents. Clin Microbiol Rev 1999;12:40-79.
yeast Candida albicans [17]. The essential oil of P. dioica showed 3. Masoko P, Eloff JN. The diversity of antifungal compounds of six
significant activity against all the test microorganisms. The workers South African Terminalia species (Combretaceae) determined
attributed the activity of the oil to its eugenol content. by bioautography. Afr J Biotech 2005;4:1425-31.
4. Newman DJ, Cragg GM, Snader KM. Natural products as sources
Zabka et al. [18] assessed the antifungal activity of essential oils of
of new drugs over the period 1981–2002. J Nat Prod
25 medicinal plants, including that of P. dioica, against two species
each of Fusarium, Penicillium and Aspergillus. The essential oil of P. 2003;66:1022–37.
dioica showed 100% inhibition of mycelial growth of all the test 5. Wagner WL, Herbst DR, Sohmer SH. Manual of the flowering
fungi. Further, the Minimal Inhibitory Concentration of the oil was plants of Hawaii. 2 vols. Honolulu: Bishop Museum Special
reported in the range of 0.5-0.7 µl/ml. They also analyzed the Publication; 1999.
essential oil by GC-MS and found that it was comprised of a number 6. Seidemann J. World Spice Plants. Heidelberg: Springer Verlag;
of compounds of which the major ones were eugenol, methyl 2005. p. 286-7.
eugenol, and β-caryophyllene. From the literature survey, it is evident 7. Sharma R. Medicinal plants of India. An encyclopedia. New
that the present study is the first report on the antifungal activity of the Delhi: Daya Publishing House; 2003.
extracts of the leaves of P. dioica against human pathogenic fungi. The 8. Jirovetz L, Buchbauer G, Stoilova I, Krastanov A, Stoyanova A,
activity of the petroleum ether extract was comparable to that of the Schmidt E. Spice plants: chemical composition and antioxidant
standard antifungal drug Miconazole. This opens up the possibility of properties of Pimenta Lindl. essential oils, part 1: Pimenta
isolating and characterizing bioactive compounds possessing antifungal dioica (L.) Merr. leaf oil from Jamaica. Nutrition 2007;31:55-63.
action against these pathogens. The test fungi used for the study are the 9. Dabur R, Gupta A, Mandal TK, Singh DD, Bajpai V, Gurav AM, et
cause of a large number of illnesses like thrush, Candidiasis, Tinea corpis al. Antimicrobial activity of some Indian medicinal plants.
and many other skin infections. Most of these are difficult to eradicate Afr J Tradit Complementary Altern Med 2007;4:313-8.
and have prolonged treatment durations with associated toxicities of the 10. Cos P, Vlietinck A, Berghe DV, Maes L. Anti-infective potential of
existing antifungal drugs. Hence new drug from a plant source could be a natural products: how to develop a stronger in vitro ‘proof-of-
source of novel, effective and less toxic curative.
concept’. J Ethnopharmacol 2006;106:290-302.
CONCLUSION 11. Bauer AW, Kirby WM, Sherris JC, Turck M. Antibiotic
susceptibility testing by a standardized single disk method. Am
The study has analyzed the antifungal potential of the extracts of the J Clin Pathol 1966;45:493–6.
leaves of Pimenta dioica, and it was found to have significant activity 12. Trease GE, Evans WC. Pharmacognosy. 11th ed. London:
against the test human pathogenic fungi comprising of yeast and
Bailliere Tindall; 1989.
dermatophyte forms. The petroleum ether extract which was
subjected to phytochemical analysis, TLC and bioautography 13. Sofowora A. Medicinal plants and traditional medicine in Africa.
revealed that the extract possessed steroids/triterpenes as the Ibadan, Nigeria: Spectrum Books Ltd; 1993. p. 191-289.
active constituents. Further studies are needed to identify and 14. Harborne JB. Phytochemical Methods. London: Chapman and
characterize the structure of the bioactive compound responsible for Hall Ltd; 1973.
the antifungal activity. The study has laid the foundation for 15. Al-Rehaily AJ, Al-Said MS, Al-Yahya MA, Mossa JS, Rafatullah S.
discovering a potent phytochemical with possibly lower side effects Ethnopharmacological studies on Allspice (Pimenta dioica) in
to treat human fungal infections. laboratory animals. Pharm Biol 2002;40:200-5.
16. Lai PK, Roy J. Antimicrobial and chemopreventive properties of
ACKNOWLEDGEMENT
herbs and spices. Curr Med Chem 2004;11:1451-60.
The authors wish to acknowledge the financial support provided by 17. Gochev VK, Girova TD. Antimicrobial activity of various
the Institution of Excellence program of the University of Mysore essential oils against spoilage and pathogenic microorganisms
(MHRD-UGC) and the Vision Group of Science and Technology, Govt. isolated from meat products. Biotechnol 2009;23:900-4.
of Karnataka. 18. Zabka M, Pavela R, Slezakova L. Antifungal effect of Pimenta
dioica essential oil against dangerous pathogenic and
CONFLICT OF INTERESTS
toxicogenic fungi. Ind Crops Prod 2009;30:250–3.
Declared none
How to cite this article
REFERENCES
Akhilesh Bhat, Raveesha KA. Antifungal activity of Pimenta dioica
1. Fisher-Hoch SP, Hutwagner L. Opportunistic candidiasis; an (L.) Merril an aromatic medicinal tree. Int J Pharm Pharm Sci
epidemic of the 1980’s. Clin Infect Dis 1995;21:897-904. 2016;8(10):92-95.

95