Piper betel essential oil.

An Invitro Study Of Determination Of Anti-Bacterial, Antioxidant, Anti- Inflammatory

Potential Of Piper Betel Essential Oil
Bhargava Paridhi*, Uppoor .S. Ashita**, Pralhad Swati**, Naik.G.Dilip**
* B.D.S.,** M.D.S, Department of Periodontology , Manipal College of Dental Sciences, Mangalore ,Manipal University ,Karnataka ,India.
Abstract: Background: Piper betel Linn is considered to possess important medicinal values. Leaves are
considered more valuable part and was used in past for preventing halitosis. Essential oil obtained from leaves
have been tried for antibacterial potential against various oral bacteria such as Streptococcous mutans,
Actinomyces species and has demonstrated effective role in suppressing plaque formation. In addition
essential oil obtained from leaves also exhibits anticancer properties because of antioxidant and anti-
inflammatory potential. However available literature falls short in evaluating antibacterial potential of Piper
betel essential oil against common periodontal pathogens- Aggregatibacter actinomycetemcomitans,
Porphyromonas gingivalis, Prevotella intermedia. Present invitro study was conducted to evaluate antibacterial
potential of Piper betel essential oil against previously mentioned periodontal pathogens by determination of
minimum inhibitory concentrations (M.I.C).Antioxidant and Anti-inflammatory potential was also determined
for the same. Methodology: Antibacterial potential was determined using Disc diffusion test and Broth
Microdilution method. Antioxidant and anti-inflammatory potential was determined by measurement of
superoxide dismutase (SOD) activity using riboflavin-NBT assay and detection of MMP-2 and MMP-9 by gelatin
zymography method respectively. Results: Piper betel essential oil is an effective antibacterial activity against
the tested periodontal pathogens along with antioxidant and anti-inflammatory potential. Conclusion: Piper
betel essential oil possesses effective antibacterial, antioxidant and anti-inflammatory potential and could be
used effectively in formulation of oral health care product. [Uppoor A NJIRM 2015; 6(2): 37-44]
Key Words: Antibacterial, Antioxidant, Anti-inflammatory, Piper betel essential oil.
Author for correspondence: Ashita S.Uppoor, M.D.S, Department of Periodontology , Manipal College of
Dental Sciences, Mangalore ,Manipal University ,Karnataka ,India. E- mail: [email protected]
Introduction: Current research targeting sources are being explored extensively as
microbial biofilm inhibition has attracted a great alternative agents in oral care products5.
deal of attention. The search for effective
antimicrobial agents against the oral pathogens Since antiquity, betel leaves (Piper betel Linn) are
such as Streptococcus mutans, Porphyromonas the most valued plant (betel vine) part and in the
gingivalis, Prevotella intermedia, Aggregatibacter past were routinely used as a chewing agent to
actinomycetemcomitans has led to identification of prevent halitosis6. The prevention of halitosis could
new agents for the prevention of oral biofilm be explained by reduction of volatility of methyl
associated diseases 1. Oral diseases such as dental mercaptan7,8 .Apart from this it is also possess
caries and periodontal diseases are caused by antibacterial, antifungal activity and causes
dental plaque which exists in a state of biofilm. increased flow of saliva9.All above properties could
be attributed due to presence of aromatic volatile
Use of antimicrobial agents along with mechanical oil containing phenolic compounds mainly chavicol
plaque control is proved to be effective in and allylpyrocatechol. Phenols and terpenes also
preventing and treating periodontal diseases2. Due contribute to the aroma of betel6.
to side effects associated with use of chemicals for
combating growth of dental biofilm, numerous Irrespective of these uses, betel vine is considered
researches have been carried to obtain to be the most maligned plant. This infamous
antimicrobial properties from plants as a suitable accreditation is principally due to the fact that
3
oral health care agent . A variety of plant materials habitual chewing of betel quid consisting of areca
and phytochemicals, especially a class of essential nut, betel leaf, catechu, slaked lime, and often
oils, have long been found to exhibit effective tobacco causes oral cancer10.
antibacterial activity against these oral pathogens4.
The aromatic molecules derived from natural But in contrast to this various scientific studies
have shown that tobacco11,areca nut12 and slaked

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 Piper betel essential oil.

lime13,14 present in the betel quid promoted Bacterial strains and growth conditions: The
carcinogenesis and betel leaf is devoid of bacterial strains that were used were Prevotella
mutagenic and carcinogenic effect. In addition intermedia (ATCC 25611), Porphyromonas
animal studies have shown conclusively that betel gingivalis (ATCC 381) and Aggregatibacter
leaf and its phytochemicals namely chavibetol, actinomycetemcomitans (JP2). All bacteria were
chavicol, hydroxychavicol, estragole, eugenol, grown in brain–heart infusion broth supplemented
methyl eugenol, hydroxycatechol prevented with 5 µg/mL of haemin and 1 µg/mL of
chemical induced cancers in experimental animals menadione (BHI with supplements), at 37⁰C under
by several mechanism such as free radical anaerobic conditions (80% N2, 10% H2 and 10%
scavenging, antioxidant, induction of detoxification CO2).
enzymes, inhibition of lipid peroxidation, anti-
inflammatory, anti-mutagenic, antitumor- The disc diffusion test and broth micro dilution
promoting and induction of selective apoptosis and tests were done to obtain the susceptibility and the
cell death of neoplastic cells15. minimum inhibitory concentration of the piper
betel essential oil.
Also aqueous extract of Piper betel oil has been
shown to inhibit plaque formation by interfering Disc Diffusion test: Brain heart infusion agar after
with adherence and acid production of early bringing to the room temperature was poured into
colonizers namely Streptococcus mutans and sterile petridish. It was then inoculated with the
Actinomyces viscosus species thus exerting anti microbial cell suspension (broth) of above
cariogenic effect3,5,16. But it should be noted that mentioned organism using a swab. The turbidity of
dental caries is a supra gingival condition, in the broth was adjusted to that of a 0.5 Mac Farland
contrast to periodontal diseases (gingivitis, turbidity standard.
periodontitis) which are sub gingival conditions
that have been linked to anaerobic Gram-negative Excess inoculum from the suspension was removed
bacteria such as Porphyromonas gingivalis, using a sterile cotton swab that was dipped and
Actinobacillus sp., Prevotella sp. and rotated against the wall of the liquid. To ensure an
17
Fusobacterium sp. . Available literature falls short even distribution, the surface of the agar plate was
in explaining potential of Piper betel essential oil swabbed three times and the plates were rotated
against periodontal pathogens (Actinobacillus sp, approximately 60 degree. Hitting sides of
Prevotella sp., Porphyromonas sp.). precipitates was avoided as it creates aerosols. The
inoculated plates were allowed to stand for atleast
Thus in this invitro study antioxidant, anti- 3 minutes, but not longer than 15 minutes before
inflammatory, antibacterial potential of Piper betel making the wells. To make wells, a hollow tube of
essential oil against Porphyromonas gingivalis, 5mm diameter was pressed on the inoculated agar
Aggregatibacter actinomycetemcomitans plate. 5 wells on each plate were similarly made.
,Prevotella intermedia which are established Each of these wells was then inoculated with 20 µl
pathogens in periodontal disease was determined Piper betel essential oil in the following
in perspective of formulating an oral hygiene concentrations (neat, 1:2, 1:4, 1:8 and 1:16). The
product to combat dental biofilm. plates were then incubated within 15 minutes of
adding Piper betel oil for 18 – 24 hours at 37 ºC in
Material and Methods: Piper betel essential oil: incubator.
Pure Piper betel essential oil was obtained from
Natural Product Company, Rym exports, Mumbai, Broth Microdilution: For broth microdilution
India. The company is registered with Basic thioglycollate broth was used as the medium. Nine
Chemicals, Pharmaceuticals & Cosmetics Export dilutions of Piper betel oil were made to estimate
Promotion Council (CHEMEXCIL), Ministry of the minimum inhibitory concentrations. First to
Commerce, Govt.Of India. Service Users of World 380µl of thioglycollate broth, 20 µl of Piper betel oil
Trade Centre, Bombay and India Indonesia was added. This was considered as the initial tube.
Business Association.

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 Piper betel essential oil.

For dilutions, only 200 µl of Thioglycollate was  The reaction mixture was then moved to a foil-
added in the nine tubes separately. lined box and illuminated with a 25W light tube
for 15 min.
For dilution, 200 µl was transferred from the initial  The inhibition of NBT reduction was
tube to the tube containing 200 µl Thioglycollate determined at measuring the absorbance at
broth. This was considered as 10-1 dilution. From 560 nm by a micro titre plate reader.
the 10-1 dilution tube, 200 µl of broth was  A negative control (water instead of the
transferred to the 2nd tube to make it 10-2 dilution. sample), positive control, and blank (addition
The serial dilution was repeated upto 10-9 dilution. of water instead of the riboflavin solution)
From the maintained stock cultures of the were evaluated at the same time per micro
microorganisms, 5 µl was mixed with 2 ml of titre plate.
Thioglycollate broth, and this suspension was  In addition, a set of standards were evaluated
added to each of the serially diluted tubes. The and these were then used to determine the
tubes were then incubated for 48 – 72 hours in an SOD activity in each test sample where SOD
anaerobic jar at 37 º C and then observed for activity was defined as that 1 unit is the
turbidity. amount of enzyme that provides a 50%
inhibition of the riboflavin-mediated reduction
Antioxidant potential: Antioxidant potential of of NBT.
piper betel oil was determined by measurement of
superoxide dismutase (SOD) activity. To determine Anti – inflammatory potential: The anti –
the SOD activity, the riboflavin-NBT assay was inflammatory potential of Piper betel essential oil
adapted from Lai18. was determined by detection of MMP-2 and MMP-
9 using Gelatin Zymography. This procedure
This method makes use of a substrate consisting of requires the preparation of 10% resolving gel
nitro blue tetrazolium chloride which reacts with (10ml) and 5% stacking gel (5ml).The sample of
superoxide ions produced upon illumination of betel leaf essential oil was prepared with addition
riboflavin in the presence of methionine’s an of 5ml of tris buffer; centrifuging at 3000rpm for 15
electron donor to produce a blue coloured min, and storing in -200C for further use. Before
complex called Formosan. experiment the sample was centrifuged at 3000
rpm for 10-15min; supernatant formed was then
The SOD present in the sample acts on superoxide used after mixing equal volume of 2x non reducing
anion produced by riboflavin and thereby reduces buffer into sample supernatant. It was mixed and
the net superoxide anions in the substrate leading pipetted into wells using gel loading tips. For
to decreased production of Formosan manifested control 50µl of betel leaf oil was pre incubated
by decreased intensity of blue colour formed. with 50µl of tetracycline (300µg/ml) for 60min at
The decrease in formation of Formosan is directly room temperature. 20 µl of test sample in each
proportional to the amount of SOD in sample, 50% well and 10 µl molecular weight marker in last well
decrease in the formation of Formosan is taken as were loaded. After this electrodes were connected.
one unit of SOD. Lid was kept on tank and plug was cabled into
power supply. The apparatus was run at about 50V
Method: for 15 min and then 100V until the bromophenol
 The test sample- betel leaf essential oil (0.1 blue reached at the bottom of the plates. After
mL) at different protein concentrations was electrophoresis was completed the gel was washed
first mixed with 2.75 mL of 67 mM phosphate with zymogram renaturing buffer i.e.2.5%Triton x-
buffer (pH 7.8) containing 0.01 M EDTA and 0.1 100 for one hour to remove SDS (Sodium dodecyl
mL of 1.5 mM NBT. sulphate) from the gel and allow proteins to
 After incubation at 37 ⁰C for 5 min, 0.05 mL of denature. The gel was then incubated in zymogram
1.2 mM riboflavin was added. at 370C overnight in zymogram incubation buffer.
Gel was then stained with Coomassie blue R-250
for one hour, after which gels were destained with
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 Piper betel essential oil.

an appropriate destaining solution for about 2 The zones of inhibition determined for Prevotella
hours. Appearance of white bands indicated the intermedia was 25mm; 23 mm at neat and 1: 2
presence of gelatinases. The lower bands present concentrations respectively; for remaining
are gelatinases-A (MMP-2) which is about 72KD concentrations it was resistant. For
while the upper bands are gelatinases-B (MMP-9) Porphyromonas gingivalis the zone was 24 mm; 22
which runs at about 95KD. mm at neat and 1:2 concentrations respectively
and resistant for other concentrations.
Results: Antibacterial activity:
The zone of inhibition representing the Table 2 summarizes the result of broth
antimicrobial activity of Piper betel essential oil at microdilution for MIC against tested
various concentrations against Prevotella microrganisms. All the three tested microrganisms
intermedia, Porphyromonas gingivalis, were sensitive to Piper betel essential oil at neat &
Aggregatibacter actinomycetemcomitans is 1:2 concentrations. They showed resistance at
summarised in Table 1. The zones of inhibition other concentrations.
were measured in mm.
Table 2: Broth Microdilution For MIC
Table 1: Zone Of Inhibitions Of Antibacterial NEAT 1:2 1:4 1:8 1:16
Activity
Neat 1:2 1: 1: 1:1 Prevotella intermedia S S R R R
4 8 6
Porphyromonas S S R R R
Prevotella 25m 23m R R R
gingivalis
intermedia m m
Aggregatibacter S S R R R
Porphyromonas 24m 22m R R R
actinomycetemcomitans
gingivalis m m
S= Sensitive R= Resistant
Aggregatibacter 24m 23m R R R
actinomycetemcom m m
Table 3: Antioxidant Activity
itans
Standard Absorbance Concentration
R= resistant
/Sample in nm in %
Aggregatibacter actinomycetemcomitans showed a
zone of inhibition of 24mm and 23 mm at neat and 1 Standard 0 500 0
1:2 concentrations respectively while being 2 Standard 1 697 10
resistant for other concentrations. (Figure 1) 3 Standard 2 707 25
4 Standard 3 720 80
Figure : 1 5 Standard 4 750 100
6 Piper betel oil 566 8.12
‘
Figure : 2

Table 3 condenses the result of antioxidant

potential of Piper betel essential oil. The different

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 Piper betel essential oil.

standard concentrations are the different

concentrations of ascorbic acid, and these Different commercially available chemical agents
concentrations of ascorbic acid have been such as cetylpyridinium chloride, chlorhexidine,
compared with the Piper betel oil depending on its amine fluorides can alter oral microbiota and have
absorbance. As an anti- oxidant, Piper betel oil undesirable side-effects such as vomiting, diarrhea
showed 8.12% activity with absorbance at 566 nm. and tooth staining, presence of ethanol (in
(Figure 2) mouthwash) have been linked to oral cancer17.

Table 4: Anti-Inflammatory Potential Natural phytochemicals isolated from plants used

Sample % of Anti- in traditional medicine are considered as good
band inflammatory alternatives to synthetic chemicals. Natural
activity products in form of essential oils are in great
1 Piper betel oil 15% 85% demand for oral health care owing to their
2 Positive Control 5% 95% extensive biological properties and bioactive
3 Negative 98% - components which have proved to be useful
Control against large number of diseases17.

Table 4 reviews the result of anti- inflammatory Piper betel oil contains large quantity of sterols
potential of betel leaf oil, wherein Piper betel oil which is the bioactive molecule responsible for
showed appearance of 15% of band. After antibacterial activity. Sterols present in the extract
calculation for appearance of band width and interact with the bacterial cell wall and membrane
thickness this 15% band corresponded for 85% altering the primary structure of cell wall and
anti-inflammatory activity while positive control membrane which ultimately lead to pore formation
showed 95% anti-inflammatory activity. (Figure 3) and degradation of the bacterial components. As
reported earlier Piper betel extracts containing
Figure: 3 high concentration of fatty acids like palmitic acid,
stearic acid and hydroxy fatty acid esters exhibits
potent antimicrobial activity against diverse
pathogenic microorganisms19. High concentrations
of flavonoids and polyphenols especially
hydroxychavicol present in Piper betel oil exhibits
potent antibacterial activity against oral cavity
pathogens5,9,19.

Various studies have evaluated antibacterial

efficacy of Piper betel oil against common oral
pathogens. The cariostatic potential of aqueous
extract of Piper betel oil was evaluated by A.R.
Fathilah et al 200316 and Nurhayati Bt Mohd Zain
20113. While the former showed this property in
Discussion: There is a global need for alternative
an invitro study by reduction in adherence activity
prevention and treatment options and products for
of early plaque settlers namely Strep. mitis, Strep.
oral diseases that are safe, effective and
sanguinis and Actinomyces sp. due to modification
economical. Available treatment strategies are
in complementary binding sites, the later showed
limited by rise in disease incidence (particularly in
downregulation of Streptococcus mutans gene
developing countries), increased resistance by
involved in attachment and plaque formation in
pathogenic bacteria to currently used antibiotics
response to treatment with Piper bêtel aqueous
and chemotherapeutics, opportunistic infections in
extract .
immunocompromized individuals and financial
considerations in developing countries17.

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 Piper betel essential oil.

The present invitro study demonstrated that Apart from these, present study also exhibited
Aggregatibacter actinomycetemcomitans, anti-inflammatory potential of Piper betel oil apart
Prevotella intermedia and Porphyromaonas from antibacterial and antioxidant property. This is
gingivalis, which are established in accordance with finding of Sharma et al 20095.
periodontopathogens were sensitive to Piper betel They found strong anti-inflammatory activity of
oil at concentrations of neat and 1:2. This indicates hydroxychavicol, measured by the estimation of
that Piper betel oil can be tried as an alternative intracellular tumor necrosis factor alpha (TNF-α)
adjunct to chemical agents against periodontal expression in a gated population of neutrophil.
pathogens.
According to Pradhan et al 201320 the methanolic
In addition, Piper betel oil was shown to have extract of betel leaf decrease the antibody titre
potent antioxidant activity because of presence of and increase suppression of inflammation thus
polyphenol compounds like catechol, suggesting possible immunosuppressive effect of
20
allylpyrocatecol by Pradhan et al 2013 and Nair et extract on cellular and humoral response. Although
al 201221 by using ascorbic acid and BHT as it should be noted that previous studies evaluated
standards. They demonstrated that betel leaf different properties of Piper betel using either
extract inhibited the radiation induced lipid aqueous or alcoholic extracts, but in the present
peroxidation process effectively because of its study these were evaluated using Piper betel
ability to scavenge free radicals involved in essential oil which is considered to be more potent
initiation and propagation steps. The extract in action as it presents as a concentrated
reduced most of Fe3+ ions and possesses strong hydrophobic liquid with various volatile aromatic
reductive ability. The extract also showed strong compounds.
hydroxyl radical and superoxide anion radical
scavenging. This can make Piper betel oil effective One of the limitation of present study includes
in treatment of medical conditions associated with determination of M.I.C only in planktonic state of
oxidative damage such as Alzheimer’s disease and mentioned microorganism (Aggregatibacter
cancer. actinomycetemcomitans,Porphyromonas gingivalis,
Prevotella intermedia),but M.I.C remains to be
Piper betel extract was also shown to reduce determined in biofilm as bacteria in oral cavity are
cellular ageing of human diploid fibroblasts by protected by biofilm, where they are less
reducing senescence-associated β-galactosidase susceptible to antimicrobial agents than their
expression, catalase activities and SOD activity by planktonic counterparts5.Thus, future studies could
Makpol et al. 201322. Using lipid peroxidation be directed to evaluate antibacterial efficacy of
Sharma et al 20095also found significant anti- Piper betel oil in an invivo model.
oxidant activity of hydroxyl chavicol present in
Piper betel extract. Hence within the limitations of present study it
could be concluded that Piper betel oil
However for determination of antioxidant demonstrated effective antibacterial activity
potential present study involved use of riboflavin – against periodontal pathogens namely Prevotella
NBT assay (by SOD activity) which was relatively intermedia, Porphyromonas gingivalis and
easy in comparison to that involved in Sharma et al Aggregatibacter actinomycetemcomitans, along
20095 as the later required rat liver microsomes with antioxidant and anti-inflammatory activity.
for determination of anti- oxidant potential. Also With the presence of all these properties the
riboflavin –NBT assay has virtue of being readily essential oil obtained from leaf could be used as an
standardized and independent of other enzymes active pharmaceutical ingredient in formulating
and proteins, such as xanthine oxidase and oral care product as an effective agent against
cytochrome c23. The result of present study periodontal pathogens.
corroborates with the finding of previous studies
thus indicating antioxidant potential of Piper betel Although further studies are still warranted for
oil. evaluation of antibacterial efficacy of Piper betel oil

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 Piper betel essential oil.

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Conflict of interest: None

Funding: None
Cite this Article as: Bhargava P, Uppoor A,
Pralhad S, Naik D, An Invitro Study Of
Determination Of Anti-Bacterial, Antioxidant,
Anti- Inflammatory Potential Of Piper Betel
Essential Oil. Natl J Integr Res Med 2015; 6 (2):
37-44

NJIRM 2015; Vol. 6(2). March –April eISSN: 0975-9840 pISSN: 2230 - 9969 44