Biochemical profiling of antifungal activity of betel leaf
(Piper betle L.) extract and its significance in traditional
Sarika Pawar1#, Vidya Kalyankar2, Bela Dhamangaonkar1, Sharada Dagade3,
Shobha Waghmode2* and Abhishek Cukkemane1
1Bijasu Agri Research Laboratory LLP, Sr. No. 37, Kondhwa Industrial Estate, Khadi Machine Chowk, Kondhwa, Pune-411048, India
2Department of Chemistry, M. E. S Abasaheb Garware College, Pune - 411 007, Maharashtra, India
3Department of Chemistry, Y. M. College, Bharathi Vidyapeeth, Pune-411038, Maharashtra, India
#Present Address: Microbial Diversity Research Centre, Dr. D. Y. Patil Biotechnology and Bioinformatics Institute, Dr. D. Y. Patil Vidyapeeth, Tathawade, Pune-
ShobhaWaghmode, Department of Chemistry, M. E. S Abasahaeb garware College, Pune - 411 007, Maharashtra, India; Tel. No:+91-9422-31-6985; E-mail:
Received: 13 February, 2017; Accepted: 10 March, 2017; Published: 20 March, 2017
Pawar S, Kalyankar V, Dhamangaonkar B, Dagade S, Waghmode S, et al. (2017) Biochemical profiling of antifungal activityof Betel leaf (Piper betle L.)
extract and its significance in traditional medicine. J Adv Res Biotech 2(1): 1-4. DOI: http://dx.doi.org/10.15226/2475-4714/2/1/00116
Piper betle (Linn) commonly called as betel leaf is a widely
cultivated plant in the Indian subcontinent. The traditional Indian
ayurvedic document describes several of its medicinal properties
including as an effective antifungal agent. The present study was
conducted to evaluate the secondary metabolite that contributes to
its antifungal activity. Invitro studies were performed on molds and
yeasts on antifungal using fractions obtained from ethyl acetate,
hexane and ethanol-methanol extracts by well-diffusion technique.
Ethyl acetate extracts showed highest anti-fungal activity. Using
biophysical techniques such as Nuclear Magnetic Resonance and
Fourier transform infrared spectroscopy techniques; we identified the
molecule as derivative of the phenyl propanoid family akin eugenol.
The molecule can be readily purified using a 2 step solvent extraction
procedure along with silica column chromatography. These findings
reveal the antifungal and possible commercial potential of the plant
extract and its potential in agriculture against pest management and
Aim: To determine the antifungal activity of Piper betle L. extracts.
Keywords: Antifungal; Ayurveda; Fourier transform infra-red
spectroscopy; Nuclear magnetic resonance spectroscopy; Food
In recent years, the increase in resistance in known fungal
pathogens to the available antifungal drugs has raised enormous
challenges to public health issues [1-3]. In addition, conventional
antifungal drugs have undesirable side effects and are very
toxic such as chlorhexidine, imidazole and amphotericin B .
One important demand of critical significance in this context is
to search for novel antifungal agents that would be less toxic
and more effective. Interestingly; several medicinal plants have
been extensively investigated in order to find novel bioactive
compounds . Moreover, several studies have suggested that
a number of plant species possesses promising antimicrobial
compounds [6-9]. Piper betle Linn. (Piperaceae), a slender
creeping plant, is widely distributed in India, Sri Lanka, Thailand
and other tropical countries. This plant has deep green heart
shaped, smooth, shinning and long stalked leaves, with pointed
apex. Betel leaf possess strong aromatic flavor and have been
long in use for the preparation of traditional Indian ayurvedic
herbal remedies. It has been reported for the treatment of various
diseases such as conjunctivitis, boils and abscesses, cuts and
injuries etc. [5,10]. In addition, it also acts as a breath freshener, a
digestive and pancreatic lipase stimulant and a pain killer in joint
pain [11,13]. Even though all these positive effects of betel leaf
are known, the biochemicals of these favorable effects remain
obscure. Moreover, betel leaf extract has previously been shown
to have strong antimicrobial effects in review [14-19]. A couple
of research articles [20,21] have demonstrated the potential of
the leaf extract on dermatophytes. Here in, we show that the
leaf extract possess antifungal activity against various plant
pathogens. The present study was sought to investigate the
effects of ethyl acetate, hexane and ethanol-methanol extracts of
this plant leave on fungal pathogens.
Here in, we employed biophysical techniques to identify
the active metabolite that provide the betel leaf extract with its
anti-fungal and other pharmacological properties that have been
valued in traditional Indian ayurveda.
Materials and methods
Betel leaf extract
The crude extract and isolation was done using Silica Gel
(100-200 mesh) for column chromatography and HPLC grade
solvents. Betel leaf was collected from Western Maharashtra farm
(Indapur tehsil), India. It was cleaned first with distilled water
and dried in shadow for a week. The dried leaves were powdered
and 100 g was used for extraction. The powder was transferred
into 1 L conical flask with 500 mL ethanol to completely soak the
powder, which was incubated at room temperature for 24 hours.
The sample was filtered using ordinary filter paper directly into
the clean round bottom flask and set for distillation. We obtained
up to 30 mL of distillate from a single run under controlled heating
at 55 ° C under reduced pressure. We repeated the procedure 3
times and pulled the collected distillate containing crude extract.
The crude extract was concentrated on a water bath at 55 ° C
under reduced pressure.
During the extraction optimization step, we added 100 mL
ethyl acetate/ethanol-methanol/ hexane to the concentrated
100 mL crude extract and mixed well. The mixture was separate
during separating funnel and the ethyl acetate/n-hexane fraction
was collected in another conical flask. All 3 extracts were
concentrated in procedure mentioned above and was further
purified using column chromatography using the respective
solvent .The eluent was pooled and analyzed by thin-layered
chromatography (TLC), Silica gel 60 F254, preloaded Silica gel on
Alumina sheets with ethyl acetate as the solvent and observed
under UV chamber and Iodine vapors.
Fungal strains and growth conditions
We tested the effect of betel leaf extract on opportunistic
fungal pathogen Aspergillus niger, which is the causative agent of
black mold in several fruits and vegetables . We also isolated
saprophytic and opportunistic pathogenic fungi Rhizopus sp.
 from the leaves of Murraya koenigii. (curry leaves). Lastly,
we isolated a mold from the leaf of figs infested with rust, which
was not obligate parasite Cerotelium (Physopella) fici  but
we have tentatively identified it as wild Aspergillus sp. These
cultures were cultured and maintained on Potato Dextrose Agar
(PDA- HIMEDIA) at 4 ° C. A stock inoculums spore suspension of
each fungal culture was prepared from fresh, mature (3-daysold)
cultures grown on potato dextrose agar plates at 28 ° C.
In-vitro antifungal assay
Antifungal activity of betel leaf extract was tested against
A.niger, wild Aspergillus sp. and Rhizopus sp. of the ethyl acetate,
hexane and ethanol-methanol extracts of betel leaf sample was
tested by well diffusion method. In brief, 500 μL of fungal spore
suspension was added to 20 mL PDA medium and poured in petri
dish. After solidification, wells of 5mm in diameter were made on
this plate. Each well was filled with 50 μL of ethyl acetate, hexane
and ethanol-methanol herbal extract. Potassium tellurite was
used as positive control and ethyl acetate, hexane and ethanolmethanol
solvent used as negative control. The antifungal assay
plates were incubated at 30 ° C for 36h. The antifungal activities
of the extracts were determined by measuring the diameter of
the inhibition zone in millimetres (mm).
NMR and FTIR
The IR spectra of neat sample were recorded on Nicolette
iD5, Thermo scientific at room temperature. Standard1 H NMR
spectra was recorded on Jeol 200MHz using CDCl3solvent and
TMS (Euriso-top) as a reference at room temperature.
Isolation of active ingredient from Piper betle extract
The beetle leaf extract was purified to homogeneity. During
the solvent extraction steps, we observed three spots on the
TLC plates. After solvent extraction the crude extract was
concentrated and run on a column containing 100-200 mesh
Silica Gel and Sodium Sulfate for further purification. In all three
solvent types that we tested we observed a single spot from there
Antifungal susceptibility assay
In the next step, we studied the effect of the organic extract
against a range of bacterial and fungal cultures. In all tested cases,
we observed antimicrobial effect of the extract against fungal
cultures only. This is in stark contrast to preciously reported
data where few groups have observed anti-bacterial effect of the
Piper betle extract [14,17,25,26]. Results obtained in the present
study revealed that the ethyl acetate extract possess effective
antifungal activity against all the tested fungal cultures (Table-1,
Figure-1).The highest antifungal activity of ethyl acetate extract
was observed for A.niger and Black rust followed by Rhizopus
sp. Antifungal activity of hexane extract was also significant
against A.niger (5 mm) and Aspergillus sp. (8 mm), while ethanolmethanol
extract was ineffective against any of the tested fungal
cultures. In addition to the molds, we also tested the effects of wild
yeasts that were isolated from leaves. We again noticed a strong
anti-fungal activity against them. From all the preparations, ethyl
acetate fraction provided the best results. This could be because
the extraction process is more efficient and perhaps also stable
over other solvent.
Biophysical characterization of the extract
We performed NMR and FTIR spectroscopy in order to
understand the functional groups associated with the isolated
active compound. In the 1H-NMR spectrum, we observed three
Inhibition zone (mm)
K-tellurite (+ control)
Antifungal activity of ethyl acetate, hexane and ethanol-methanol
herbal extract against indicated fungi.
Figure 1: Antifungal activity of ethyl acetate, hexane and ethanol-methanol
extracts of herbal sample against A) A.niger B) Aspergillus sp. and
C) Rhizopus. Extract preparation in a) Hexane, b) Ethanol-Methanol, c)
Ethyl acetate and positive control d) Potassium tellurite.
types downfield peaks at 6.7, 7.2 and 7.25 ppm for aromatic
ring. Additionally plenty of peaks were observed for protons
associated with polar and aliphatic groups in the 3.25-5.5
and 1-2.25 ppm, respectively. From the characteristics of the
spectra and the results published by other groups, the extracted
compound is a derivative of the phenyl propanoid family to
which antimicrobials eugenol-chavicol belong . The FTIR
spectra well corroborated with the NMR spectrum. We observed
a broad peak in the range of 3700 to 3000, which corresponds
to absorption caused by N-H, C-H and O-H single bonds. We
did not observe any peaks characteristic of triple bonds in the
range of 2,500 to 2,000. A wide range of double bond specific
groups in C=O and C=C was observed in the region from 2,000 to
1,500. Lastly, the region from 1700-600 show finger print that is
reminiscent of phenyl propanoid eugenol [27,28].
The antifungal activity of the various extract highlights that
ethyl acetate based extraction process provided the best for
antifungal property. Surprisingly, unlike other results where
Piperbetle extract that showed both anti- bacterial and fungal
activities, we did not observe any anti-bacterial effect against
gram positive Staphylococcus aureus and Bacillus sp.; few
gram negative bacteria such as Escherichia coli, Pseudomonas
aeruginosa and Xanthomonas campestris. This may be due to
the fact that our purification method is different to the ones
reported before (Table 2). In all previous studies crude extracts
have been used hence many groups have reported anti-bacterial,
anti-fungal and in some cases anti-cancer effects as well. In
our methodology, we have enriched the phenyl propoanoid
derivative in the extract by first ethanolic extract and then
treating with other organic solvents like ethyl acetate/ hexane/
Figure 2: 1D 1H-NMR spectrum of the active ingredient of Piper betle
extract is suggestive of molecule from the phenyl propanoid- eugenol
Figure 3: FTIR spectrum of the active ingredient of Piper betle extract.
anti-fungal (Ali et al., 2010);
antibacterial (Sharma et al 2009 )
anti-bacterial (Basak and Guha, 2015)
Concoction- boiling extraction
anti-cancer (Gundala et al., 2014)
ethanol or water- soxhlet extraction
Streptococcus mutans (Deshpande and Kadam, 2013)
solvent extraction: methanol, ethyl acetate and petroleum ether
Streptococcus mutans (Deshpande and Kadam, 2015)
anti-bacterial (Sugumaran et al., 2011)
Hydroxychavicol and Eugenol
anti-cancer (Paranjpe et al., 2013)
liquid-liquid and supercritical fluid extraction
Comparison of extraction methods(Singtongratana et al., 2013)
Summary of bio-active extraction procedures from Piper betle
ethanol-methanol. Furthermore, the large zone of clearance that
we observe highlights the better extraction and stability of the
phenyl propanoid in the ethyl acetate fraction.
Our finding sheds light on one of the important biochemical
metabolite that contributes to the significance of betel leaf in
traditional Indian ayurvedic medicine. The metabolite can be
readily extracted in a two-step process using organic solvents
and silica column chromatography. The active ingredient
belongs to the phenyl propanoid family belonging to eugenolchavicol
group have been shown to possess strong antimicrobial
properties [15,26,29,30]. More importantly, our results highlight
the potential of using betel leaf extract as a potent anti-fungal
agent for farming and perhaps also food storage against different
types of molds. Food spoilage is a major agricultural problem
accounting for heavy losses; therefore it is necessary to make
the process commercially viable and efficacious against various
pathogens and food spoilage organisms [31,32].
We thank the following institutes like. M. E. S AbasahebGarware
college, Vijay Chemicals and Neeti Developer for support; and Dr.
Nivedita Cukkemane for critically reading the manuscript
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