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Piper betle L.: A review on its ethnobotany, phytochemistry, pharmacological profile


and profiling by new hyphenated technique DART-MS (Direct Analysis in Real Time
Mass Spectromet...

Article  in  Journal of Pharmacy Research · October 2011

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Kushagra Nagori et al. / Journal of Pharmacy Research 2011,4(9),2991-2997
Review Article Available online through
ISSN: 0974-6943 www.jpronline.info
Piper betle L.: A review on its ethnobotany, phytochemistry, pharmacological profile and profiling by
new hyphenated technique DART-MS (Direct Analysis in Real Time Mass Spectrometry).
Kushagra Nagori*, Mukesh Kumar Singh, Amit Alexander, Tekeshwar Kumar, Dhansay Dewangan, Hemant Badwaik, D.K. Tripathi
Rungta College of Pharmaceutical Sciences and Research, Bhilai 490006, Chhattisgarh, India.
Received on: 19-05-2011; Revised on: 08-06-2011; Accepted on:01-07-2011

ABSTRACT
The Piper betle plant is an evergreen and perennial creeper which is used in several traditional medicines to cure various diseases. This plant has been known
to possess antioxidant, antifungal, antiulcerogenic, antiplatelet, antidiabetic, immunomodulatory, antileishmanial, antiamoebic, anti-inflammatory, antifilarial
and antimicrobial activity. A wide range of chemical compounds in cluding chavibetol, allyl pyrocatechol, eugenol, quercetin, caryophyllene, safrole,
hydroxychavicol, a-pinene, myrcene, chavicol, Germacrene-D, a- terpineol , ß-pinene, camphene etc have been isolated from this plant. The present review
summarizes the information concerning the botany, ethnopharmacology, phytochemistry, biological activity and use of hyphenated analytical technique like
DART-MS (Direct Analysis in Real Time Mass Spectrometry) and other techniques for characterizing various compounds from P. betle plant.

Key words: P.betle, DART-MS, creeper, antioxidant, chavibetol.


INTRODUCTION
Occurrence, Botanical description and Ethnopharmacology Earlier gastro protective properties of the leaf extract were reported on ex-
From the ancient time, man has been using plants as medicine. From a histori- perimentally induced gastric lesions13. A phenolic compound hydroxychavicol
cal perspective, it has been evident that the fascination with plants is as old as with anticarcinogenic property has also been identified in betel leaves14. Piper
mankind. Herbs have provided us some of the very important life saving drugs betle is not recommended as a antidiabetic agent in traditional medicinal sys-
used in the armamentarium of modern medicine. The plant kingdom repre- tems. However it is possible that Piper betle leaves may posses antidiabetic
sents a rich source of organic components, many of which have been used for properties as Piper sarmentosum and Piper longum close relatives of the
medicinal & other purposes. Herbal medicines remain the major source of plant, have been shown to have antidiabetic properties 15, 16. The use of P. betle
health care for the world’s population 1. Currently, there is a growing interest L. leaves to lighten melasma may produce leukomelanosis, which manifests as
in plant based or herbal medicines even in the western world. In many re-
confetti-like skin depigmentation 17; also the roots (8-12g) are used in treating
spects, the mechanism of action of the herbal drugs differs from that of the
synthetic drugs or pure compounds2. In one of the study of the World Health rheumatism 6. The leaf produces an aromatic volatile oil containing a phenol
Organization it is estimated that 80% of the population of developing coun- called chavicol which has powerful antiseptic properties. Piper betle inflores-
tries relies on traditional plant based medicines for their health requirements3. cence contains high levels of safrole (15mg/g wet weight) and chewing betel
Piper betle Linn. (Betle vine) is a tropical plant closely related to the common quid containing Piper betle inflorescence generates a high concentration of
pepper and belongs to the family Piperaceae 4. It is extensively grown in India, safrole (420 µM) 19,20. The plant possesses cardiotonic 41 wound healing3 9
Srilanka, Malaysia, Thailand, Taiwan and other Southeast Asian countries and chemopreventive 53 and reversible antifertility80 activities. Table 1.
has a long history of over 2000 yrs44. The stems are dichotomous, articulate, Table 1 Ethnomedicinal importance of Piper betle.
swollen and rooted at nodes 3mm in diameter, woody and with 2.5 to 4cm long Sr.No. Plant part used Ethnomedicinal Use
internodes. Stem stout with pinkish-stripe along node dilated and rooting5.
The leaves are simple, spiral and exstipulate. The petiole is 5mm long, chan- 1. Whole Plant The liquid extract of the plant has been used traditionally in curing inflam-
mation and infection of the respiratory tract, cough, dyspnoea, indigestion,
neled and pubescent. The blade is 10 x 6cm & 9.5 x 5cm, ovate to ovate – diphtheria, hysteria as well as general and sexual debility 21 . A concoction of
oblong and light green below. The base of the blade is cordate and the apex is indigenous Indian drugs containing P. betle dry extract was found to be an
acuminate, the secondary nerves are in three pairs. The inflorescence is an effective long-lasting oral contraceptive 23 . Piper betle is also reported to
possess antifungal, antiseptic and anthelmintic properties to serve as a con-
axillary spike which is 5.5 cm long. The fruits are drupaceous, orange, and traceptive for humans and to possess antihypertensive properties. 30, 31 The
3mm in diameter 11. The betel leaf is known as Paan (Assamese, Urdu, Hindi, users believe that chewing the ‘paan’ improves their efficiency and stamina32
Odia, Bengali) and Bhakshyapatra, bhujangalata, bhujangavalli, divabhishta, . Piper betle showed hypotensive, cardiac, and respiratory depressant ef-
kalaskanda, nagavalli, nagavallika, nagini, parna, vitika (Sanskrit). Some of fects smooth and skeletal muscles relaxant actions, antimicrobial, fungicidal,
and nematocidal activity 33-35 . Medicinally P. betle is an aromatic, carmina-
the names in the regions in which it is consumed are: betel leaf vine (English), tive, stimulant and astringent used as a preventive for worms and in snake bite
Vetrilai (Tamil), Tamalapaku (Telugu), Vidyache pan (Marathi), Veeleyada 36
.
yele (Kannada), Vettila (Malayalam), Plu (Mon), Malus (Tetum), Maluu 2. Leaves
Betel leaf has been described from ancient times as an aromatic, stimulo-
(Khmer), Plue (Thai), Bulath (Sinhalese), Malu (Tokodede), Bileiy (Divehi), carminative (katu), astringent and aphrodisiac (kamagnisandipanam) 12 .The
Bulung samat (Kapampangan language), daun sirih (Malay language), Papulu leaves are credited with wound healing37, 39 . The Indian traditional system of
medicine has identified the P.betle leaves with digestive and pancreatic li-
(Chamorro), Ikmo (Philippines) and Tr?u (Vietnamese)5,12. Ayurveda an In- pase stimulant activities.3 7 , 3 9 - 4 2 which has also been proved with experimen-
dian system of medicine has been an integral part of Indian culture and tal animals..43 Betel leaf is traditionally known to be useful for the treatment
materia medica. From the rich Indian biodiversity, it has identified various of various diseases like bad breath, boils and abscesses, conjunctivitis, con-
plants/herbs that have been associated with a number of potential therapeutic stipation, headache, hysteria, itches, mastitis, mastoiditis, leucorrhoea, otor-
rhoea, ringworm, swelling of gum, rheumatism, abrasion, cuts and injuries
efficacies2. etc as folk medicine.44 Fresh juice of betle leaves is also used in many
ayurvedic preparations. 45 Leaves are also considered useful in treating bron-
PLANT PROFILE chitis, difficulty in breathing and cough.37 Traditionally Piperbetle leaf extract
is reported to inhibit male reproductive competence. 46-48 Piper betelleaves
Kingdom: Plantae is used in eyedrops for eye injury/infection as a baby lotion for the new born,
Division: magnoliphyta for coughs, asthma, constipation and to arrest milk secretion.49 Essential oil
Class: magnolipsida from leaves of this plant has been used for the treatment of respiratory
catarrhs and as antiseptic.5 0 . Piper betle leaves are shown to posses
Order: Piperales hepatoprotective activity,51 antifertility on male rats48 and anti motility
Family: Piperaceae effects on washed human spermatozoa 46 biological activities described for
Genus: Piper the essential oil include antifungal, antiseptic and anthelmintic effects 80 .
Species: P. betle In folk medicine root is known for its female contraceptive effects44 . The roots
3. Roots are used as a contraceptive and are chewed by singers to improve their voice
*Corresponding author.
32

In ancient times flower is used as an ingredient for chewing food known as betel
4. Flower
Kushagra Nagori quid in South-East Asia .24 .
Rungta College of Pharmaceutical Sciences 5. Fruit The fruit is employed with honey as a remedy for cough 50 .
and Research, Bhilai 490006,
Chhattisgarh, India.
Tel.: + 91-09691500850, 0788-2350698
E-mail:kushagranagori13@yahoo.co.in

Journal of Pharmacy Research Vol.4.Issue 9. September 2011 2991-2997


Kushagra Nagori et al. / Journal of Pharmacy Research 2011,4(9),2991-2997
PHYTOCHEMISTRY
The chemical constituents of betel essential oil consist of mainly terpenes and
phenols53. Leaves contain protein 3.1 %, carbohydrate 6.9 %, minerals 2.3 %, and
tannins 2 %. Leaves contain bitter compounds that are about 0.7 to 2.6 %. The
nutritional composition of fresh Piper betle leaf: water (85-90%), Protein (3-
3.5%), Fat (0.4-1.0%), minerals (2.3-3.3%), Fibre (2.3%), Chlorophyll (0.01-
0.25%), Carbohydrate (0.5-6.10%), Nicotinic acid (0.63-0.89mg/100g), Vitamin
C (0.005-0.01%), Vitamin A (1.9-2.9 mg/100g), Thiamine (10-70 µg/100g), Ri-
boflavin (1.9-30 µg/100g), Tannin (0.1-1.3%), Nitrogen (2.0-7.0%), Phosphorus
(0.05-0.6%), Potassium (1.1-4.6%), Calcium (0.2-0.5%), Iron (0.005-0.007%),
Iodine (3.4 µg/100g), essential oil (0.08-0.2%, Energy (44 kcal/100g) 44. The betle
leaves have strong pungent aromatic flavor54 and the characterstic flavor of betel (3) Myrcene (C 10H 16 ) (4) Cadinene (C 15H 24)
is due to the betel phenols 55. More recent work with the leaves was found to
contain starch, diastases, sugars (0.8 to 1.8 %) and an essential oil to an extent of
4.2%, the specific gravity varies from 0.958 to 1.05712. Phytochemical investi-
gation on leaves revealed the presence of tannins and steroidal components.56.
The terpenoids include 1,8-cineole, cadinene (Fig.1.4), camphene, caryophyllene,
limonene, pinene, chavicol, allyl pyrocatechol, carvacrol (Fig.1.8), safrole, eu-
genol, and chavibetol are the major phenols found in Piper betle, there acetates
are also commonly found55,44. Eugenol was identified as the antifungal principle in
the oil.38 Some of the phytoconstituents of the plant are summarized in the Table
2.
Table. 2. Phytoconstituents of Piper betle
Active principle Plant part used Reference
(5) Camphene (C 10H 16) (6) Thujene (C 10H 16)
Chavibetol Leaves 57,22,54,58,59,60
Allyl pyrocatechol Leaves 58
Chavibetol acetate Leaves 61,62,57
Eugenol (Fig 1.1) Leaves &Flower 63,56,64
Allyl pyrocatechol Leaves 65,66,60
Quercetin Leaves &Flower 67&64
Luteolin, stearaldehyde Leaves 68, 42
a- terpineol , ß- pinene, camphene Leaves 62
Caryophyllene (Fig.1.9) leaves 57, 69,26
Safrole Leaves & Flower 63,64,70,64
Hydroxychavicol Leaves &Flower 56,71,64
ß- sitosterol Leaves & Roots 56,72,72
Diosgenin Leaves 56
?- lactone, allyl catechol , eugenol methyl ether, Leaves 73
cepharadione A, dotriacontane, triacontane
Piperine Stem 73,72
Piperlonguminine Stem 73
4-allyl resorcinol, humulene (Fig.1.17), stigmast-4-en-3,6- Roots 73 (7) Limonene (C 10H 16) (8) Carvacrol (C 10H 14O )
dione, aristololactam A-II, ß-sitosteryl palmitate, 3- ß-acetyl
ursolic acid , ursonic acid
Linalool (Fig.1.10) Leaves &Rhizome 62,22
Allyl diacetoxy benzene Leaves 38
Gallic acid, procatechuic acid, chlorogenic acid, caffeic acid, Whole plant 74
ferulic acid, ellagic acid
Isoeugenyl acetate Whole plant 69
N-Isobutyl-2E,4E-decadienamide, pipedardine, piperine Whole plant 75
4-allyl phenyl acetate ,4-allyl phenol, a- myrcene, D- Leaves 62
limonene, eucalyptol, camphor (Fig.1.15), allyl anisole, 4-allyl
phenol, ß-iso safrole (Fig.1.16), thymol (Fig.1.18), 3-allyl-6-
methoxyphenol, a-bergamotene, a-farnesene, a- muurolene, (9) Caryophyllene (C 15H 24 ) (10) Linalool (C 10H 18O )
a-bisabolene, ledol , a-cadinol, isoledene
a- tocopherol Leaves 76
Eugenyl acetate, allyl pyrocatechol diacetate, allyl Leaves 63,29
pyrocatechol monoacetate, terpinen -4-ol , thujene(Fig.1.6),
camphene (Fig.1.5), sabinene, 1,4 cineole, (Z)-ß-ocimene, (E)-
ß-ocimene, cis sabinene hydrate, fenchone, terpinolene, 2-
noanone, cis-limonene oxide, sabinene (Fig.1.14), m-cymen-
8-ol, cis-piperitol, n-decanal, thymol, 2-undecanone, iso-
ascaridole, 5-indanol, a-cubebene, a- copaene (Fig.1.10),
(E)-ß- Damascenone, vanillin , a-selinene, cuparene,
germacrene A, n-eicosane
a-pinene Leaves &Rhizome 63, 26,22
Chavicol (Fig.1.2) Leaves 63
Myrcene (Fig.1.3) Flower & Rhizome 70,63,24,22
Isoeugenol Flower 70,63,64, 62
ß- sitosteryl palmitate, dotriacontanoic acid, stearic acid, Leaves 72
cepharadione -A
(11) a- copaene (C 15H 24) (12) Germacrene D (C 15H 24 )
Methyl eugenol, flavones Flower 64
Benzene acetic acid , hexadecanoic acid, octadecanoic acid, Leaves 71
myristic acid,2,3-bis (hydroxy) propyl ester , 2 mono
palmitin, limonine (Fig.1.7), octadecanoic acid, 2,3-
bis(hydroxyl) propyl ester
Isoeugenyl acetate Leaves 69
(E)-ß-ocimene, terpinolene, allo ocimene, cymene (Fig.1.13). Leaves 27,28
ß- caryophyllene Leaves & Rhizome 77,62,22
Germacrene-D (Fig.1.12) Leaves 62

(13) Cymene (C 10H 14) (14) Sabinene (C 10H 16)


(1) Eugenol (C 10H 12O 2) (2) Chavicol (C 9H 10O )
Journal of Pharmacy Research Vol.4.Issue 9. September 2011 2991-2997
Kushagra Nagori et al. / Journal of Pharmacy Research 2011,4(9),2991-2997
which showed promising activity against obligate oral anaerobes responsible
for halitosis. The P.betle ether extract and APC could thus be used in the
prevention of halitosis in addition to the prevention of periodontal infection
caused by the colonization of oral anaerobic bacteria. The biological studies
with APC indicated that the potential to reduce methylmercaptan and hydro-
gen sulfide was mainly due to the anti-microbial activity as established using
dynamic in vitro models 65. The adhering ability of microorganisms to the
tooth surface determines the developmental stage of dental plaque. With
suitable environmental and nutrients, carcinogenic plaque will subsequently
develop. To minimize the development of carcinogenic plaque, the adhering
ability of the early settlers needs to be control problem the aqueous extract of
Piper betle is used which reduced the adherence of early plaque bacteria to an
(15) Camphor (C 10H 16O) (16) Safrole (C 10H 10O 2) experimental pellicle. The extracts were observed to have the ability to make
them less adherent. This could account for the significant reduction in the
hydrophobic binding capacity of the bacteria when treated with the extracts 81.

Antifungal activity
The investigation attempted to identify new antifungal agents by screening
two varieties of Piper betle (green vein and red vein) showed strong activity
against all the pathogens tested (Colletotrichum capsici, Fusarium
pallidoroseum, Botryodiplodia theobromae, Alternaria alternata, Penicil-
lium citrinum, Phomopsis caricae-papayae and Aspergillus niger) with inhi-
bition diameters significantly (P < 0.01) bigger than 2.5 mg/ml prochloraz or
10 mg/ml clotrimazole. The minimum inhibitory concentrations of the
ethanolic extracts of P. betle against these plant pathogens ranged between
0.01 mg/ml & 1 mg/m1 82.
(17) Humulene (C 15H 24) (18) Thymol (C 10H 14O ) Antileishmanial activity
An ethanolic extract of leaves of Piper betle (PB) was tested for its
Fig. 1 (1-18) Chemical structures of some chemical constituents iso-
antileishmanial activity that was evidenced in both promastigotes and
lated from various parts of Piper betle. amastigotes, with IC50 values of 9.8 and 5.45 µg/ml, respectively; importantly,
PHARMACOLOGICAL ACTIVITY it was accompanied by a safety index of >12-fold. This leishmanicidal activity
of PB was mediated via apoptosis as evidenced by morphological changes, loss
Antioxidant activity of mitochondrial membrane potential, in situ labeling of DNA fragments by
Oxidative damage is an important effect of ionizing radiation on biological terminal deoxyribonucleotidyl transferase mediated deoxyuridine triphosphate
membranes. It is a chain reaction. Free radicals generated from the radiolytic nick end labeling, and cell-cycle arrest at the sub-G0/G1 phase. Here it is
decomposition of water can attack fatty acid chains of membrane lipid. A free anticipated that phenolics identified in PB such as APC and chavibetol could
radical that has sufficient energy to abstract an allylic hydrogen from the contribute towards the observed antileishmanial activity.68.
methylene carbon of polyunsaturated fatty acids can initiate the peroxidative
process. Here the presence of Piper betle leaf extract inhibited the radiation Radioprotective activity
induced lipid peroxidation process effectively. This could be attributed to its The radioprotective activity of Piper betle ethanolic extract has been studied
ability to scavenge free radicals involved in initiation and propagation steps. using rat liver mitochondria and pBR 322 plasmid DNA as two model in vitro
Oral supplementation with extract (1, 5 and 10 mg/kg) was administered daily systems. The extract effectively prevented ?-ray induced lipid peroxidation as
for 2 weeks to Swiss albino mice and the hepatic antioxidant status was analysed. assessed by measuring thiobarbituric acid reactive substrates, lipid hydroper-
The GSH content was enhanced and no appreciable change was found in the oxide and conjugated diene. Likewise, it prevented radiation-induced DNA
levels of oxidative damage in terms of lipid peroxidation. Also, the specific strand breaks in a concentration dependent manner. The radioprotective ac-
activity of SOD increased in a dose dependent manner. These factors indicate tivity of PE could be attributed to its hydroxyl and superoxide radicals scav-
the elevation of antioxidant status in the animals. The effect on the glyox- enging property along with its lymphoproliferative activity. The radical scav-
alase system which is considered to be activated under stress conditions was enging capacity of PE was primarily due to its constituent phenolics, which
also investigated. Our findings did not observe any significant change in gly I were isolated and identified as chavibetol and allyl pyrocatechol 58.
and gly II activities, implying a non-stress condition after oral treatment of
the extract. The present study indicates the antioxidant activity of P. betle Antiulcerogenic activity
leaf extract and its potential to elevate the antioxidant status 79 . Further Pretreatment of an ethanolic extract of leaf of Piper betle Linn at a dose of
investigation shows that the 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay of 200mg/kg body weight, orally administered to rats for ten consecutive days,
the ethanol extracts of three varieties (Bangla, sweet, and Mysore) of Piper was found to posseses a significant protective action against gastric lesions
betle (pan) revealed the Bangla variety to possess the best antioxidant activ- induced by indomethacin. The extract pretreatment resulted in significant
ity that can be correlated with the total phenolic content and reducing powers increase in superoxide dismutase (SOD) and catalase (CAT) activity, increase
of the respective extracts. Column chromatography of the extract of the in mucus, hexosamine and total thiol group content, but marked reduction in
Bangla variety led to the isolation of chavibetol (CHV), allylpyrocatechol oxidatively damaged protein and peroxidised lipid levels as compared to un-
(APC), and their respective glucosides. The HPTLC analysis of the extracts treated ulcerated control. The extract was also found to possess both superox-
revealed similar chemical profiles in all three P. betle varieties, although the ide and hydroxyl free radical scavenging action. The present observation
concentrations of CHV and APC were significantly less in the sweet and establish the efficacy of the extract in prevention of experimentally induced
Mysore varieties. Among the isolated compounds, APC showed the best results peptic ulcer by indomethacin and antioxidant property appears to be pre-
in all the in vitro experiments. It could prevent Fe (II)-induced lipid higher dominantly responsible for such cytoprotective activity in the experimental
than 4µg/ml 78. One study investigated that chavibetol, eugenol, methyl eu- model 83. Further investigation showed the protective activity of allyl pyrocat-
genol, and peroxidation (LPO) of liposomes and rat brain homogenates as well echol (APC), which is the major antioxidant constituent of P.betle against the
as ç-ray-induced damage of pBR322 plasmid DNA more efficiently than CHV indomethacin-induced stomach ulceration in the rat model. This was done by
2
The antioxidant activity was further shown in an aqueous IPB (inflorescence treating the rat mode l with APC (2mg/kg body wt per day) and misoprostol
.
Piper betle) extract and was found to be a scavenger of H2O2, superoxide (1.43 µg/kg body wt per day) for 7 days and effectively heals the stomach
radical, and hydroxyl radical with a 50% inhibitory concentration (IC50) of ulceration as revealed from the ulcer index and histopathological studies. The
about 80, 28, and 73 µg/mL, respectively. IPB extract also prevented the healing capacities of APC and misoprostol could be attributed to their antioxi-
hydroxyl radical induced PUC18 plasmid DNA breaks at concentrations car- dant activity as well as the ability to enhance the mucin content of the gastric
vacrol played an important role in antioxidant effectiveness 79. tissues 66. Another investigation revealed that the healing activity of ethanolic
extracts of Piper betle against the indomethacin induced stomach ulceration
Treatment of halitosis has been studied and compared with that of misoprostol. It was found that the
Piper betle L. (Piperaceae) leaves which are traditionally used in India and excellent healing activity of ethanolic extract of P. betle play a major role of
China in the prevention of oral malodor was examined by bioassay-guided mucin protection and regeneration in the healing of non-steriodal anti-in-
fractionation to yield allylpyrocatechol (APC) as the major active principle flammatory drugs mediated stomach ulceration 84.

Journal of Pharmacy Research Vol.4.Issue 9. September 2011 2991-2997


Kushagra Nagori et al. / Journal of Pharmacy Research 2011,4(9),2991-2997
Antiplatelet activity for all the three life-stages for water decoction was between 1.5 and 6.25 ml/
ROS (reactive oxygen species) are crucial for platelet aggregation and IPB ml. Regarding crude methanolic extract, LC100 values ranged between 7.8 and
extract has been found to inhibit the arachidonic acid (AA) induced and col- 31.25 mg/ml. Interestingly, among the different fractions, the chloroform
lagen-induced platelet aggregation, with an IC50 of 207 and 335 µg/ml, re- fraction was the most active on adult B. malayi, showing LC100 of 3.9 mg/ml,
spectively. The IPB extract also inhibited the AA-, collagen- (>100 µg/mL of followed by hexane fraction which was effective at 31.2 mg/ml; n-butanol and
IPB), and thrombin (>250 µg/mL of IPB)-induced thromboxane B2 (TXB2) aqueous fractions were ineffective 86.
production by more than 90%. However the effect of thrombin-induced ag-
gregation was not significant. These results indicated that aqueous compo- Anti-inflammatory activity
nents of IPB are potential ROS scavengers and may prevent the platelet The study reported that the crude leaf powder suspension of P. betle was
aggregation possibly via scavenging ROS or inhibition of TXB2 production 78. evaluated for acute and chronic anti-inflammatory study at a dose of 300 mg/
kg. Diclofenac sodium was used as the standard drug. Carrageenan and dextran
Immunomodulatory activity models were studied for acute inflammation while cotton pellet induced granu-
Many of the disorders today are based on the imbalances of immunological loma was used for chronic inflammation study. ANOVA followed by Dunnett’s
processes. This necessitates the search for newer and safer immunomodulators. test were employed for statistical analysis. The study indicates that the P.betle
With this objective a study was conducted to explore the immunomodulatory exhibit significant anti inflammatory activity 85.
activity of the methanolic extract of Piper betle L. The MPb consists of
mixture of phenols, flavonoids, tannins and polysaccharides. Both in vitro as Anti-amoebic & anti-giardial activity
well as in vivo evaluation was carried out. The effects of MPb on lymphocyte The anti-amoebic activity of P.betle was studied with chloroform extract of
proliferation, interferon-C receptors and the production of nitric oxide were P.betle leaf which was commonly used by AIDS patients in southern Thailand.
measured in vitro. Further, the extract at different dose levels was studied in It was screened at a concentration of 1,000 µg/ml, against Entamoeba
vivo for the humoral and cellular immune responses on mice immunized with histolytica strain HTH-56:MUTM and strain HM1:IMSS growing in vitro.
sheep red blood cells.the result showed that P.betle significantly suppressed The extract was incubated with 2×105E. histolytica trophozoites/ml of me-
phytohaemaglutinin stimulated peripheral blood lymphocyte proliferation in dium at 37°C under anaerobic conditions for 24 h. The cultures were examined
a dose-dependent manner. The decrease in antibody titre and increased sup- with an inverted microscope and scored (1-4) according to the appearance and
pression of inflammation suggests possible immunosuppressive effect of ex- numbers of the trophozoites the extract caused inhibition and retested using
tract on cellular and humoral response in mice. The study indicated the poten- the same conditions but with concentrations that ranged from 31.25 to 1,000
tial of MPb as a novel candidate for immunosuppressive activity. The same µg/ml using E.histolytica strain HM1:IMSS, and the IC50 values for each ex-
could be further evaluated for its anticancer activity or as a potential candi- tract was calculated. The chloroform extract of P.betle with IC50 value of 55.2
date in the treatment of autoimmune disorders such as rheumatoid arthritis, µg/ml was classified as active i.e. with an IC50 of less than 100 µg/ml. The IC50
systemic lupus erythomatous or emphysema 56. of a standard drug metronidazole was 1.1 µg/ml 87. Also the study investigated
the anti-giardial activity of chloroform extract of P.betle leaf which was
Antimicrobial activity commonly used as self medication by AIDS patients in southern Thailand. The
The methanolic and aqueous extracts showed strong activity against the yeasts: standard drug metronidazole and plant extract was incubated with 2 x 105
C. albicans, and M. pachydermatis. The crude essential oil exhibited a broad- trophozoites of Giardia intestinalis per millilitre of growth medium in 96-well
spectrum strong antimicrobial activity against all test organisms. The stron- tissue culture plates under anaerobic conditions for 24 h. The chloroform
gest activity was observed against C. albicans, followed by S. aureus and M. extract of P.betle was shown to be active i.e. with an IC50 of 100 < µg/ml. the
pachydermatis62. Further an investigation reported the antimicrobial activity results shows that the MIC & IC50 values of P.betle leaf have potential use
of crude aqueous extract of P. betle on Streptococcus mutans. Transmission against infection caused by G. intestinalis 88.
electron microscopy (TEM) was used to determine the effect of the extract
on the ultrastructure of S. mutans. The study showed cells with nucleoid ANALYTICAL TECHNIQUES USED IN IDENTIFICATION OF CHEMI-
material coagulated in to thick electron dense filaments and destruction of the CAL CONSTITUENTS OF P. BETLE
plasma cell membrane and inner cell wall. The effect is more significant with Profiling of Piper betle Linn. by Direct Analysis in Real Time Mass
the higher concentration of the extract. The effect of crude extract on the Spectrometric Technique.
ultra structure of S. mutans observed in this study could be due to the fatty Identification and quantitation of bioactive components from raw materials
acids and hydroxyl fatty acid ester components present. The hydrophobic and processed products has been routinely performed by HPLC-PAD, HPLC-
parts of the compound may enable them to partition the lipids of the bacterial MS,GC-MS, etc. Although such methods have been successfully adopted to
cell membrane, thereby disturbing the structures and rendering them more various fields of natural products and crude herbal drugs, there is an increasing
permeable. When the membrane is more permeable, other components present requirement for more efficient and prompt analytical techniques in order to
in the extract could make its way in to the bacterium and coagulate the manage vast numbers of samples. The quick and efficient analysis and real
nucleoid while maintaining the cell intact. Analysis of the effect on the acid time detection of bioactive components in raw materials and processed prod-
producing propertieswas analysed by pH drop assay. The extract was found to ucts would be one of the most important steps in facilitating the successful
significantly reduce acid producing properties of the bacteria 71. Antibacterial development of products containing botanical drugs and functional foods. In
activity of P.betle was checked against 15 clinically important bacterial strains order to evaluate the extent of the advantage offered by DART-MS in provid-
it shows moderate activity against B.cereus. Here cefotaxime (100µg/disc) was ing real time analysis of natural products from raw materials, a pilot study was
used as a standard. From results it can be said that P. betle can be explored performed with the well known anti-oxidant botanical drug Piper betle 94 . The
further as they are able to resist bacterial growth up to some extent 85. new technique referred to as Direct Analysis in Real Time has been coupled to
the AcuuTOF-LC atmospheric pressure ionization mass spectrometer to per-
Mosquito larvicidal and Tyrosinase inhibition activity mit high-resolution, exact mass measurements of gases, liquids, and solids 96.
While mosquito larvicidal activity was carried out with the essential oil and Using a helium plasma DART ionizes atmospheric water and generates water
methanolic and aqueous extracts of P. betle. The essential oil exhibited the clusters which in turn ionize the sample held in the gas stream. The resulting
larvicidal activity significantly with 2 h and 24 h, LD50 value of 86 and 48 spectra are relatively clean and simple. As DART ion source can ionize mol-
ppm, respectively. The methanolic extract of P. betle showed larvicidal activ- ecules directly from the surface plant products, which can be analyzed directly
ity with 2 h and 24h with LD 50 value of 153 and 125 ppm, respectively, without sample preparation 97, 98, 99. The eight varieties of Piper betle leaves
whereas the aqueous extract showed slight activity. The study suggested that were analysed by DART-MS named Bangla, Desawari, Deshi, Jaleshar green,
the essential oil and methanolic extract of P. betle is a potential natural Jaleshar white, Kalkatiya, Mahoba and Saufia. The results were shown in the
mosquito larvicide 62. Inhibition of mushroom tyrosinase by the essential oil, Table 3.
methanolic and aqueous extracts of P. betle was evaluated. The essential oil
exhibited concentration-dependent inhibition of mushroom tyrosinase, giv- Table .3. Exact mass data from the DART mass spectra of Piper betle leaves
ing an IC50 value of 126 ppm. The essential oil was fractionated into two Molecular Measured Calculated Molecular Error Remarks
fractions. The fraction I showed a strong inhibition of tyrosinase activity weight mass mass formula (mmu)
concentration-dependently, at an IC50 value of 115 ppm. The presence of the
134 135.08128 135.08099 C9 H11 O 0.29 Chavicol
hydroxyl group at the 4 position of the aromatic ring (4-allylphenol, eugenol) 150 151.07640 151.07590 C9 H10 O2 0.50 Allylpyrocatechol
in the essential oil, may play an important role in the inhibition of tyrosinase 164 165.09242 165.09155 C10 H13 O2 0.87 Chavibetol
62 165 166.08864 166.08680 C9 H12 NO2 0.84 Phenyl alanine
.
174 175.07764 175.07590 C11 H11 O2 1.74 Unknown
176 177.09109 177.09155 C11 H13 O2 -0.46 Chavicol acetate
Antifilarial activity 192 193.08772 193.08647 C11 H13 O3 1.25 Allylpyrocatechol
Both water decoction and crude methanol extract of BM in general appeared acetate
206 207.10140 207.10212 C12 H15 O3 -0.72 Chavibetol acetate
to possess good antifilarial efficacy against all the life-stages of B. malayi, 251 252.12287 252.12358 C13 H18 NO4 -0.71 Unknown
viz. adult worms, vector derived infective larvae and microfilariae. The LC100
Journal of Pharmacy Research Vol.4.Issue 9. September 2011 2991-2997
Kushagra Nagori et al. / Journal of Pharmacy Research 2011,4(9),2991-2997
Table No. 4 Chemical constituents obtained by different analytical techniques in P.betle.
P. betle varieties Part used Identification method used Active principle Reference

Indian (Saufia, Bangla, Leaves DART-MS Chavicol, allylpyrocatechol, chavibetol, 55


Desawari, J.Green, J.White, Phenyl alanine, chavicol acetate, chavibetol acetate,
Kalkatiya, Mahoba, Deshi Paan ) allylpyrocatechol acetate, allylpyrocatechol diacetate.
Vietnamese Rhizome oil Combination of capillary GC, a- cadinol, d- cadinene. 22
GC-MS & 13 C-NMR.
Leaves GC & GC-MS Isoeugenol and isoeugenyl acetate 89
Taiwanese Flowers GC/MS Safrole (28%), & myrcene (26%) 24
HPLC and TLC Safrole, hydroxychavicol, eugenol, isoeugenol, eugenol methyl ether.
S. marcescens treated betlevine Leaves and roots HPLC Gallic,, protocatechuic, chlorogenic, caffeic, ferulic, ellagic acid. 74
P.betle Herba Whole plant TLC, HPLC-DAD, GC-MS Piperdardine, piperine. 75
—— Leaves GLC Safrole, eugenol, allyl pyrocatechol diacetate, methyl chavicol,terpineol. 91
Betle inflorescence Water extract HPLC Safrole (78.9%), eugenol (6.2%) 92
Srilankan Leaves GC-MS Safrole, allyl pyrocatechol diacetate, eugenol & eugenol acetate 63
Indian (South) Leaves GC-FID, GC-MS, olfatometry. Chavibetol, eugenol, allyl pyro catechol derivatives. 93
Indian Leaves GC, GC-MS ß - caryophyllene (less than 26.5% ) 77
Indian (Bangla) Leaves Column Chromatography Chavibetol, allyl pyrocatechol 60
Malasian Leaves GC-MS Hydroxychavicol,hexadecanamide,2-monopalmitin, hexadecanoic acid, 2,3- 71
bis(hydroxy)propyl ester, myristic acid, 2,3-bis(hydroxy)propyl ester, benzeneacetic
acid, alpha-hydroxy phenyl.

The mass spectrometer used was a JMS-100 TLC (AccuTof) atmospheric pressure 7. Tobacco Habits Other than Smoking; Betel Quid and Areca-nut Chewing and Some
ionization time-of-flight mass spectrometer (Jeol, Tokyo, Japan) fitted with a Related Nitrosamines. IARC Monographs on the Evaluation of the Carcinogenic
DART ion source. The mass spectrometer was operated in positive-ion mode with Risk of Chemicals to Humans, (IARC Press: Lyon) 1985, 37.
a resolving power of 6000 (full-width at half-maximum).The orifice 1 potential 8. Sankaranarayanan R, Oral cancer in India: An epidemiological and clinical
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potentials were set to 13 and 5 V, respectively. Orifice 1 was set to a temperature 9. Moore S R, Johnson N.W, Pierce A.M, and Wilson D.E., The epidemiology of mouth
of 100°C. The RF ion guide potential was 300 V. The DART ion source was cancer: a review of global incidence, Oral Diseases, 6, 2000, 65–74.
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set to 300°C. The potential on the discharge needle electrode of the DART source Dental Association 70(4), 2004, 244–248.
was set to 3000 V; electrode 1was 100 V and the gridwas at 250 V. Freshly cut 11. Wiart C, Medicinal plants of Asia & Pacific, (Pub-CRC Press) Taylor & Francis
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spectrometer for measurements. Data acquisition was from m/z 10 to 1050. Exact 12. Chopra R.N, Chopra I C, Indigenous Drugs of India. 2 nd Edition, (Pub- Academic
mass calibration was accomplished by including a mass spectrum of neat polyeth- Publishers) 1958, 372.
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rate to within_0.002 u. Using the Mass Center software, the elemental composi- novel role of free radical scavenging action. Indian J. Exp. Biol,. 41, 2003, 311-315.
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are shown in Table 4.
17. Bour-Jr Wang, Yue-Liang Guo, How-Ran Guo, Ho-Yuan Chang, Piper betle L. inflo-
CONCLUSION rescence causes allergic contact dermatitis of the hands during betle quid assembly.
The scientific research on P. betle suggests a huge biological potential of this Contact dermatitis 58, 2008, 368–370.
plant. It is strongly believed that detailed information as presented in this 18. Yu-Ting Chung, Chiu-Lan Chen, Cheng-ChungWu, Shan-An Chan, Chin-Wen Chi,
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Journal of Pharmacy Research Vol.4.Issue 9. September 2011 2991-2997

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