PHCOG REV.

REVIEW ARTICLE

Investigations on Piper betle grown in Sri Lanka

Arambewela L. S. R., Arawwawala L. D. A. M., Kumaratunga K. G1, Dissanayake D. S, Ratnasooriya W. D. 2,
Kumarasingha S. P.3
Industrial Technology Institute, Bauddhaloka Mawatha, Colombo 7, 1Food Control Laboratory, Ministry of Health, Anuradhapura, 2Department of
Zoology, University of Colombo, 3Colombo North Teaching Hospital, Ragama, Sri Lanka (currently at Royal Perth Hospital, Perth, Australia)

Submitted: 07-07-2010 Revised: 18-09-2011 Published: 23-12-2011

ABSTRACT
Piper betle is an economically important plant cultivated in Sri Lanka. Although more than 12 cultivars of betel are reported
in Sri Lanka, very few scientific investigations have been carried out on them. Studies on the chemical constituents indicated
that safrole is the major constituent, followed by chavibitol acetate, in the essential oil of common betel leaves of Sri Lanka.
Investigations on the bioactivities of P. betle revealed the presence of antimicrobial, insecticidal, antioxidant, antinociceptive,
antidiabetic and gastroprotective activities. In addition, P. betle was found to be safe in terms of hepatotoxicity, renotoxicity,
hematotoxicity, gross morphology, weights of organs, stress or aversive behaviors in rats. The above findings indicate the
vast potential of P. betle yet to be harnessed for the benefit of mankind and the betel industry of Sri Lanka.

Key words: Anatomy, bioactivities, chemical constituents, morphology, Piper betle, safety profile

INTRODUCTION major buyer of betel, has reduced the volume of betel imported
from Sri Lanka.[4]
Piper betle Linn. (Sinhala name: Bulath, English name: Betel)
belongs to the genus Piper of the plant family Piperaceae.[1] Although betel vine has been cultivated in Sri Lanka for
Over 700 species of plants belonging to the genus Piper are centuries, very few research activities have been carried out
on it, except studies on antiaphrodisiac activity,[5] antifertility
distributed in both hemispheres.[2] Of these, about 30 species
effects on male rats[6] and antimotility effects on washed human
have been reported from India.[1] In Sri Lanka, 18 species of
spermatozoa.[7] However, P. betle grown in other countries has
genus Piper are found; and out of them, three are endemic.[3]
been shown to possess antimicrobial,[8] gastroprotective,[9] wound
healing,[10] hepatoprotectve[11] and antioxidant[12] activities. In
Betel leaves have been traditionally used for chewing purposes
order to minimize the negative impact on the betel industry in
along with other condiments. Colombo, Gampaha, Kalutara, Sri Lanka, there is a necessity to study important bioactivities of
Kurunegala, Kegalle, Ratnapura, Matale and Galle are the main Sri Lanka grown betel leaves and develop value added products
betel-cultivating districts in the country. In addition to a wide based on these activities. This strategy will safeguard both
and well spread domestic market, betel has gained a significant growers and the economy of the country. In order to achieve this,
position in the export market since 1974. Betel industry, at times, possible bioactivities of betel oil and extracts were investigated
faces severe problems of depressed prices and restricted export using betel leaves from Sri Lanka. In addition, morphological,
market. The main cause for this situation is that Pakistan, our anatomical and chemical studies were also conducted. These
investigations and their results are summarized below.
Address for correspondence:
Dr. Menuka Arawwawala
Industrial Technology Institute, Bauddhaloka Mawatha, Colombo
7, Sri Lanka. E-mail: [email protected]/
CLASSIFICATION
[email protected]
Kingdom: Plantae
Access this article online Order: Piperales
Quick Response Code:
Family: Piperaceae
Website: Genus: Piper
www.phcogrev.com Species: P. betle

Morphological and anatomical studies of betel

DOI:
10.4103/0973-7847.91111 Six cultivars of P. betle Linn., namely, Galdalu, Mahamaneru,
Kudamaneru, Ratadalu, Nagawalli and Malabulath, were used

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 Arambewela, et al.: Piper betle and bio-activities

in the present study.[13] Morphological and anatomical features, ANTIMICROBIAL SCREENING STUDIES
including parameters such as stomatal index, leaf length-to-width
ratio, were similar in Kudamaneru, Mahamaneru, Galdalu, Ratadalu Antibacterial activity
and Nagawalli but were different in the cultivar Malabulath. In the present study, the EO from the leaves showed activity
against Escherichia coli, Streptococcus pyogenes and Staphylococcus aureus.
The Minimum Inhibitory Concentration (MIC) values were 3.12
CHEMICAL CONSTITUENTS AND × 102, 2.50 × 103 and 5.00 × 103 μg/mL, respectively. The ethanol
PHYSICOCHEMICAL PROPERTIES OF THE extract showed activity against Streptococcus pyogenes, Escherichia coli
ESSENTIAL OIL and Staphylococcus aureus. The MIC values were 1.25 × 103, 5.00
× 103 and 5.00 × 103 μg/mL, respectively.[13, 14]
Chemical composition
According to the chemical constituents present in the essential Antifungal study
oil (EO), cultivars of Galdalu, Mahamaneru, Kudamaneru, This assay revealed that EO of P. betle contained at least three
Ratadalu and Nagawalli were similar and they were categorized fungicidal compounds, and the ethanol extract contained at
under “common betel.” EOs from the leaves, stalks, stems, least one fungicidal compound active against Cladosporium sp.
fruits and roots of common betel and from the leaves of Further, antifungal activity of betel oil was investigated against
Malabulath were also analyzed.[13] Major constituents of the Colletotrichum sp., Fusarium oxysporium sp., Corynospora cassicola,
Rigidoporous sp. and Phytophthora sp., using the disk method. All
EO of common betel were found to be safrole (48.7%) and
fungi species except Phytophthora sp. showed significant growth
chavibitol acetate (12.5%). Malabulath does not contain these
inhibition in betel oil.[13, 14]
two compounds. The major compound in Malabulath oil is
allylpyrocatechol diacetate (34.0%), which is the third major
compound in common betel oil (11.3%). Further, p-cymene, INSECTICIDAL ACTIVITIES
4-terpineol, safrole, eugenol, β-caryophellene and chavibitol
acetate detected in common betel leaf oil were not detected Mosquito larvicidal assay
in Malabulath leaf oil. The Gas Chromatography-Mass Late 3rd instar larvae of Aedes egeptii were introduced into P.
Spectroscopy analysis of the EO of different parts of common betle EO at 500, 100, 50, 25, 12.5, 6.25 ppm concentrations,
betel indicated that composition of the stalk oil was different to and mortalities were recorded after 1 hour and 24 hours.
that of the other parts, as it did not contain detectable amounts Mortalities of 43% and 100% were observed for 100 and 500
of allylpyrocatechol diacetate. The major compound detected ppm concentrations, respectively, within 1 hour. Compared to
in the EO from the leaf, the stem, the stalk and the root was the control, significant mortality was observed even at lower
safrole; but in the fruit oil, it was β-phellandrene. This chemical concentrations, 25 and 50 ppm, after 24 hours.[14]
composition of the EO of leaves appears to be closer to that
Bioassay for housefly (Musca domestica)
of cultivar Deshawari in India.
In this assay, betel EO (120 μg/cm3 in ethanol) showed 100%
knock down effect and mortality against Musca domestica.[14]
The composition of the betel EO changes with the maturity
of the leaf. It was observed that the contents of major
Bioassay for rice weevil (Sitophilus oryzae)
compounds safrole and chavibitol acetate in the leaf were at
Betel EO at 1%, 0.8% and 0.5% concentrations was used in
the maximum level at the harvesting stage. Moreover, eugenol
this study. Mortality rate of 100% was observed in 1% betel oil
and β-phellandrene content decreased with maturity, and solution within 1½ hours.[14]
β-phellandrene content remained constant after maturity. Allyl
pyrocatechol diacetate content increased up to the harvesting Bioassay for Chrysomya megacephala larvae
stage and remained constant thereafter. The study on the Betel EO solutions ranging in concentration from 1% to 4%
variation of the composition of the EO with maturity is useful were prepared using 1% Tween 80, sodium lauryl sulfate (0.05
in deciding the maturity stage at which the leaf has to be collected g per 100 mL, as a stabilizer) and methyl paraben (0.01 g per
for applications that depend on specific compounds. Further, it 100 mL, as a preservative). The 4% and 3% preparations of the
justifies why Ayurvedic physicians mention the maturity of the oil of betel were effective in killing 100% of the larvae of C.
plant in drug preparations. megacephala within 3½ hours, while betel oil at 2% concentration
killed 97% of C. megacephala larvae within 4 hours. The positive
Physicochemical properties control, mineral turpentine, also killed the larvae within 4
The physicochemical properties of the EOs of Kudamaneru, hours. This shows that betel oil is effective in the treatment
Mahamaneru, Galdalu, Ratadalu and Nagawalli too were of wound myiasis.[15]
similar but were different from those of Malabulath. These
studies indicate that physicochemical properties and chemical Bioassay for Chrysomya bezziana larvae
constituents of the EO of Malabulath are different from those A study was conducted to evaluate the efficacy of betel EO
of other cultivars. against the larvae of Chrysomya bezziana in vitro.[10] With 4% betel

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 Arambewela, et al.: Piper betle and bio-activities

oil, all 1st instar larvae were killed within 2 hours, and the 2nd ANTIDIABETIC ACTIVITY
instar larvae were killed within 4 hours. The positive control
(Asuntol) showed no mortality until 4 hours, but all larvae were Overall results show that both HWE and CEE of P. betle leaves
weak - from first 30 minutes. In the negative control, larvae were from Sri Lanka possess marked hypoglycemic activity (when tested
mobile and active. Betel oil at 3% killed all the 1st instar larvae in fasted normoglycemic rats) and antihyperglycemic activity
within 150 minutes; and 74% of the 2nd instar larvae, within 4 (judged by improvement in the results of glucose tolerance test and
hours. These results indicate that betel oil from Sri Lanka is an by lowering of the blood glucose levels in rats with streptozotocin
effective larvicide for C. bezziana 1st and 2nd instar larvae in vitro. (STZ)-induced diabetes).[18] The hypoglycemic effect of P. betle
extracts (100, 200, 300 mg/kg) on fasted normoglycemic rats was
dose dependent and lasted up to 4 hours, except that of the lowest
ANTIOXIDANT ACTIVITY
dose of HWE. Further, hypoglycemic potential of HWE and CEE,
respectively, was comparable to that of tolbutamide, the reference
The extracts obtained from the leaves of P. betle had profound
hypoglycemic drug of the sulphonylurea type.
antioxidant activity as judged by Thiobarbartiuric Acid Reactive
Substances (TBARS) and 2, 2 – diphenyl – 1 – picrylhydrazyl
In glucose tolerance test, HWE, CEE and tolbutamide lowered
(DPPH) scavenging assays.[14,17] The scavenging effects of P. betle
the external glucose level in a similar manner. Further, HWE
extracts on DPPH radicals increased in the following order: Cold
significantly reduced the blood glucose level of rats with
Ethanolic Extract (CEE) > EO > HWE. Further, free radical
STZ-induced diabetes treated with a dose (50 mg/kg) which
scavenging effect of CEE was higher than that of synthetic
is known to irreversibly damage the insulin-secreting β cells of
antioxidant Butylated Hydroxy Toluene (BHT). Employing
the pancreas. This suggests that an intact-endocrine pancreas
the DPPH assay, Indian researchers[12] have investigated the
and insulin are not essential for antidiabetic activity of P.
antioxidant activities of three betel cultivars (Kauri, Ghanagete
betle extracts. This ability of lowering the blood glucose levels
and Bagerhati) grown in India. The antioxidant activities of
of rats with STZ-induced diabetes also suggests that P. betle
the three cultivars were in the order of Kauri > Ghanagete >
extracts have insulinomimetic activity. It is possible that HWE
Bagerhati, but the free radical scavenging ability of commercial
may act as an insulin secretagogue and/or sensitize insulin
betel from Sri Lanka is higher than that of the reported Indian
receptors, as proposed for some plant extracts. HWE failed to
cultivars.
significantly inhibit glucose absorption from the lumen of the
intestine. However, HWE provoked accumulation of glycogen
In TBARS assay, the antioxidant potential of CEE was the best
in the liver and the skeletal muscle. This is another peripheral
among the P. betle extracts tested, and this effect was significantly
mechanism through which HWE exhibits its antidiabetic activity.
higher than that of BHT and green tea, respectively. Compared
This increased glycogenesis may result from enhanced glucose
to EO and HWE, the degree of delaying the lipid peroxidation
uptake from liver and skeletal muscle by sensitization of insulin
was significantly lower in BHT and in green tea. Safrole is the
receptors and/or inducing the activity of enzymes involved in
major constituent in the EO of Sri Lankan commercial betel
glycogen synthesis.
leaves. However, compared to the EO, the antioxidant activity
of safrole was significantly low. This suggests that antioxidant
potential of EO is not only due to safrole but possibly due to GASTROPROTECTIVE ACTIVITY
synergetic effect of all volatile constituents.
A study to evaluate the gastroprotective activity of HWE and
Interestingly, the antioxidant properties of P. betle extracts, CEE of P. betle leaves was carried out.[19] Three doses (200,
including CEE, EO and HWE, remained unaltered for a period 300 and 500 mg/kg) of both the extracts were evaluated for
of 12 months at room temperature (as evaluated by DPPH gastroprotective activity against ethanol-induced gastric ulcers
assay). This supports the potential use of the betel extracts as in rats. Oral administration of HWE and CEE provided marked
a natural antioxidant in food industry. However, when P. betle dose-dependent and significant protection against gastric damage
extracts were exposed to elevated temperature (200°C), the caused by absolute ethanol. The gastroprotective effect of HWE
antioxidant property was significantly reduced (CEE and EO by was comparable to that of CEE. Further, the gastroprotective
4 fold; HWE by 3 fold). Similar results were also evident with activities of the highest dose of both extracts were significantly
the synthetic antioxidant BHT (by 4 fold). Even after exposure greater than the gastroprotective activity of misoprostol, the
to the elevated temperature, the antioxidant potential of CEE reference drug. A similar study conducted using Indian betel
was higher than that of BHT. cultivars has been reported.[9] In this experiment, treatment
with ethanol extract of betel leaves at a dose of 150 mg/kg for
In an attempt to introduce betel as a natural antioxidant, the CEE 10 consecutive days produced significant healing effect in rats
was incorporated into fats (cake margarine), oils (coconut and palm with ulcers induced by nonsteroidal anti-inflammatory drugs
oil) separately and its rancidity determined in terms of peroxide (NSAIDs).
value (PV). The results showed that PVs were significantly lower
in CEE-treated samples than in BHT-treated samples. The HWE significantly increased the mucus content (by 49%)

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 Arambewela, et al.: Piper betle and bio-activities

adhering to the wall of the gastric mucosa. Mucus layer is controls. Furthermore, neither extract of P. betle induced any
considered to be important in the mucosal defense against overt signs of toxicity (salivation, diarrhea, lacrimation, tremors,
endogenous aggressors, e.g., acids, and also as an agent in ataxia, yellowing of hair, loss of hair, postural abnormalities
facilitating its repair. It is generally believed that enhanced acid or behavioral changes), stress (fur erection or exophthalmia)
secretion is the most important factor for the induction of or aversive behaviors (biting paw and penis, intense grooming
gastric lesions. In this study, the highest dose of HWE did not behavior, scratching behavior, licking at tail or vocalization).
cause significant inhibition in acidity (both total and free) or pH
of gastric fluid. Therefore, the gastroprotective effect of P. betle Neither extract significantly changed any of the serum parameters
was not mediated via inhibition of acid secretion in the gastric [aspartate aminotransferase (AST), alanine aminotransferase
mucosa but by increasing its mucus content. (ALT), urea and creatinine] and hematological [red blood cell
(RBC) counts, white blood cell (WBC) counts and hemoglobin
(Hb) concentration] parameters investigated. All the tested organs
ANTINOCICEPTIVE ACTIVITY
(liver, kidneys, testes, adrenal glands, heart, spleen, vasa deferentia,
The CEE and HWE of betel leaves have antinociceptive activity, prostate glands, seminal vesicles together with coagulating glands
as evaluated in the hot plate test and in the tail flick test in rats. and caput plus corpus epididymides) appeared normal in all treated
This indicates centrally mediated antinociceptive activity of the rats. There were no significant changes in the organ weights
plant extracts against acute pain.[20] The antinociceptive activity between the treated groups except for the spleen. Compared to
of extracts (100, 200, 300 mg/kg) was genuine, as there was no the control, in both treated groups significant increase in weight
change in the retain retention times in the Bar and Bridge tests as of the spleen was evident (CEE- 217.4%; HWE- 234.8%). Gastric
compared to controls. Both 200 and 300 mg/kg doses of P. betle lesions were not observed in any of the treated rats. In conclusion,
extracts markedly reduced the licking time in early and late phases this study shows that both cold ethanolic and hot water extracts
of the formalin test in a bell-shaped dose-response curve. In the of Sri Lankan betel leaves and leaf stalk were safe following sub-
formalin test, the pain in the early phase is caused due to the direct chronic oral administration to rats.
stimulation of the sensory nerve fibers by formalin, while the
pain in the late phase is due to the inflammatory mediators, like A similar study conducted in India also reported that ethanolic
histamine, prostaglandin, serotonin and bradykinin. It is reported extract of P. betle leaf stalks was nontoxic as judged by
that NSAIDs reduce both phases of the formalin test. The betel hematological, biochemical profiles and enzymatic studies.[22]
extracts too induced interruptions in both phases of this test,
suggesting possible impairments of sensory transmission and DEVELOPMENT OF VALUE-ADDED PRODUCTS
release of inflammatory mediators. The highest antinociceptive
activity was evident with 200 mg/kg dose of both HWE and FROM BETEL
CEE. As antinociceptive activity of CEE was higher than
that of HWE, CEE was used to investigate its antinociceptive Based on the results of scientific investigations, several
mechanism. value-added products such as betel toothpaste, mouthwash, face
cream, shampoos, instant betel quid, betel pellet, antitick lotion,
Some sedatives possess antinociceptive activity. However, antitick powder and wound healing creams were developed
the antinociceptive action of CEE is unlikely to be mediated in order to enhance the marketability of betel and improve
via sedation, as none of the parameters monitored in the Rat the prospects of the industry. Clinical trial conducted using
hole board technique were changed. Antinociception can be the wound healing cream on dermatitis patients revealed that
induced via dopaminergic mechanisms, but such a mode of treatment was significantly effective on skin rashes.[23] At present
action is unlikely in this study as metoclopramide, a dopamine a clinical study is in progress to evaluate the antidiabetic activity
receptor antagonist, failed to block antinociception induced by of spray-dried powder of betel hot water extract.
the extract. The opioid receptor, antagonist naloxone, blocked
the antinociception induced by CEE, suggesting that the
CONCLUSIONS
antinociception was mediated through opioid mechanisms.
P. betle is a common plant in Sri Lanka, and it can be easily
SAFETY PROFILE cultivated in any part of the country. This scientific study
revealed for the first time the chemical constituents and
There were no treatment-related deaths or morbidity with CEE multifaceted activities of betel cultivated in Sri Lanka. The
(1,500 mg/kg/d) and HWE (1,500 mg/kg/d) even after following Chrysomya megacephala and C. bezziana larvicidal activities of
sub-chronic (14 days) and chronic (42 days) oral treatment in betel as well as its antidiabetic and antinociceptive activities
rats. [21] Further, CEE and HWE treated rats showed normal food have been reported for the first time, herein. The fact that betel
intake, water intake, and their percentage weight gain significantly leaves have multiple activities such as antimicrobial, insecticidal,
reduced. The consistencies of feces and color of urine in CEE antioxidant, antinociceptive, gastroprotective and antidiabetic, as
and HWE treated rats were essentially similar to those of the revealed in this study, indicates that P. betle is a good candidate

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 Arambewela, et al.: Piper betle and bio-activities

for future herbal drug preparations and development. This aromatica and Piper betle. Fitoterapia 1990;61:458-9.
hitherto untapped vast potential of betel grown in Sri Lanka, if 11. Saravanan R, Prakasam A, Ramesh B, Pugalendi KV. Influence
properly harnessed, will safeguard the betel industry of Sri Lanka, of Piper betle on hepatic marker enzymes and tissue antioxidant
status in ethanol - treated wister rats. J Med Food 2002;5:197-204.
enhance the livelihood of a large number of villagers depending
12. Dasgupta N, De B. Antioxidant activity of Piper betle L. leaf
on betel industry and introduce novel herbal products and drug extract in vitro. Food Chem 2004;88:219-24.
preparations into the market.
13. Arambewela L, Kumaratunga KG, Dias K. Studies on Piper betle
of Sri Lanka. J Natn Sci Foundation Sri Lanka 2005;33:133-9.

ACKNOWLEDGMENTS 14. Chemical studies on Piper betle and development of value added
products. Final report submitted to CARP. [CARP library] (2004).
15. Kumarasingha, SP, Ihalamulla RL, Arambewela LSR,
The authors are grateful to Prof. R. L. C. Wijesundara, The
Dissanayake DS. Larvicidal effects of mineral turpentine, low
Dean, University of Colombo, for providing facilities to conduct aromatic white spirits, aqueous extracts of Cassia alata and
microbiological studies; and to Dr. H. A. Ratnasoma, Betel Research aqueous extracts, ethanolic extracts and essential oil of betel
Station, Narammala, for providing betel cultivars. Financial assistances leaf (Piper betle) on Chrysomya megacephala. Int J Dermatol
provided by Council for Agricultural Research Policy and National 2002;41:877-80.
Science Foundation are gratefully acknowledged. 16. Wardhana AH, Kumarasingha SP, Arawwawala LDAM,
Arambewela LSR. Larvicidal efficacy of essential oil of betel
leaf (Piper betle) on the larvae of the old world screwworm fly,
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Antimotility effects of Piper betle (L) leaf extract on washed S. Efficacy of betel cream on skin ailments. J Complement Integr
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Kumaratunga KG, Dissanayake DS, Ratnasooriya WD,
9. Majumdar B, Chaudhuri SG, Ray A, Bandyopadhyay SK.
Effect of ethanol extract of Piper betle Linn leaf on healing of Kumarasingha SP. Investigations on Piper betle grown in Sri
NSAID - induced experimental ulcer - A novel role of free radical Lanka. Phcog Rev 2011;5:159-63.
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Source of Support: Nil, Conflict of Interest: None declared
10. Santhanam G, Nagarajan S. Wound healing activity of Curcuma

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