ISSN: 2349-5251
journal homepage: www.ijbsans.com
Ranjithkumar Rajamani1, Selvam Kuppusamy*2, Shanmugavadivu. M1 and D. Rajmohan3
1
Department of Biotechnology, Dr.N.G.P. Arts and Science College, Coimbatore, Tamilnadu, India.
Department of Botany, Periyar University, Salem, Tamilnadu, India.
3
Department of Zoology, Kongunadu Arts and Science College, Coimbatore, Tamilnadu, India.
2
Received: Sep 2015; Accepted Jan 2016
*Author to whom corresponding should be addressed
Email: selsarat@yahoo.com
Abstract: Plant kingdom represents a massive reservoir of biologically active compounds. Around 60%
of the drugs used in medicine are of obtained from plant origin. It is important to use phytochemical
methods to screen and analyse biological active components and their therapeutic mechanisms. Areca
catechu Linn also known as areca nut is one of the stimulant masticatory crude materials of Indian
system of medicine. Piper betel L. also known as betel leaves contains large number of biomolecules
which are used for treating cough and foul smelling in mouth. In this present work demonstrated on
phytochemical screening of the aqueous extracts of betel nut and betel leaf were screened by using
standard methods. The aqueous extract were prepared at 15 min by simple technique. Preliminary
phytochemical screening of betel nut and betel leaves showed the presence of active compound in the
aqueous extract. The positive result for the presence of alkaloids, saponins, phenol, reducing sugar,
triterpinoids and glycosides substance observed in aqueous extract of betel nut. The aqueous extract of
betel leaves showed the presence of preliminary phytochemical such as alkaloids, saponins, coumarin
and glycosides substance.
Keywords: phytochemical analysis, betel nut, betel leaf, medicinal plant.
1. INTRODUCTION
Plant materials are used throughout the
world as home remedies, over the counter
drug products and raw substances for the
pharmaceutical industry, cosmetics industry
and represent a substantial proportion of the
world drug market. It is therefore significant
to establish their quality. In this study we
selected a widely available plant material
such as betel nut and betel leaf. India is one
of the largest populated countries in the
world and it has eight different geographical
zones. Recent study revealed that the Areca
catechu extract increases learning and
memory (Madhusudan et al., 2012). In rural
India, the consumption of betel nut (Areca
nut) with betel leaf is common practice.
Areca nut (Areca catechu) popularly known
as “betel nut”, is one of the oldest known
masticatories amongst Asia (Trease and
Evan, 2009). Betel nut contains numerous
medicinally active compounds it was cited
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for the medicinal properties, specially
antibacterial and antiviral activity (Reena et
al., 2009; Sastry, 2005).
Over 100 varieties of Piper betel has been
distributed in both of the hemispheres of
world of these 40 species have been recorded
in India. The parts of Piper betel utilized, are
leaves, roots, stems, stalks and fruits. The
deep green heart shaped leaves of betel vine
are popularly known as Pann, in India and
vettrilai in Tamilnadu. The leaf juice is used
for fever, cough, fatigue, asthma, to disinfect
wounds externally (Rai et al., 2011). A
preliminary study has reported Piper betel
leaves extracts contains large numbers of
bioactive molecules like polyphenols,
alkaloids, steroids, saponins and tannins
(Koff et al., 1971). Shun et al. (2007) study
indicated the leaves extract of Piper betel
exhibit
biological
capabilities
of
detoxication, anti-oxidantion and antiJ. Biosci. Nanosci., 3(1):2016
mutation that suggested the chemopreventive
potential of extracts against various ailments
including liver fibrosis and carcinoma.
Research on identification of biologically
active compound of plant origin as an
alternative remedy to various health issues is
well advanced. In the present work deals
with preliminary phytochemicals analysis of
betel nut and betel leaf aqueous extract by
using standard methods.
Triterpinoids, Phlobatannins, Acids and
Glycosides.
2.3.1 Alkaloids (Raaman, 2006): Solvent
free extract, 50 mg of the plant sample was
stirred with one ml of dilute hydrochloric
acid and filtered. The filtrate was tested for
alkaloids.
2.3.2 Mayer’s Test: To the filtrate, a drop of
Mayer’s reagent was added along the sides
of the test tube. A white precipitate indicates
the test as positive.
2.3.3 Flavonoids (Alkaline reagent test):
Two ml of aqueous solution of the extract
was treated with 1 ml of 10% ammonium
hydroxide solution. Yellow fluorescence
indicates the presence of flavonoids
(Raaman, 2006).
2.3.4 Saponins: Fifty mg of the plant sample
was ground with 3 ml of distilled water and
diluted with the same, made-up to 20 ml.
The suspension was shaken in a graduated
cylinder. After 15 min, a two cm layer of
foam indicates the presence of saponins
(Raaman, 2006).
2.3.5 Phenols (Ferric chloride test): About
50 mg of the sample was dissolved in 5 ml of
distilled water. To this few drops of neutral
5% ferric chloride solution was added. A
dark green color indicates the presence of
phenolic compounds (Raaman, 2006).
2.3.6 Glycosides (Keller-kilani test): Crude
extract was mixed with 2ml of glacial acetic
acid containing 1-2 drops of 2% solution of
FeCl3. The mixture was then poured into
another test tube containing 2ml of
concentrated H2SO4. A brown ring at the
interphase indicated the presence of cardiac
glycosides.
2.3.7 Reducing Sugar: To 0.5 ml of the
aqueous extract of the plant sample, 1 ml of
water and 5-8 drops of Fehling’s solution
was added at hot and observed for brick red
precipitate ((Iyengar, 1995).
2.3.8 Tannins: One ml of water and 1-2
drops of ferric chloride solution was
separated and 1 ml of aqueous extract of the
plant sample. Blue color was observed for
gallic tannins and green black for catecholic
tannins (Iyengar, 1995).
2. MATERIALS AND METHODS
2.1 Preparations of the betel nut extract:
Betel nut was collected from local market
nearby our college, Coimbatore, Tamilnadu,
India. The betel nuts was rinsed with
distilled water thoroughly to remove dirt and
other attached particles. The betel nuts
extract was prepared by taking 20 g of
thoroughly washed and finely cut betel nuts
in a 250 ml Erlenmeyer flask with 100 ml of
sterile distilled water and then boiled the
mixture for 10 min. The solution was then
removed from the head source and left at
room temperature. Following this step the
extract was then filtered through a Whatman
filter paper No.1. The extract was kept in
refrigerator at 4ºC for further experiments.
2.2 Preparations of the betel leaf extract:
Apparently healthy betel leaves were
collected from local market nearby our
college and washed thoroughly in tap water
to remove dirt and other attached particles.
The betel leaves extract was prepared by
taking 20 g of thoroughly washed and finely
cut betel leaf in a 250 ml Erlenmeyer flask
with 100 ml of sterile distilled water and
then boiled the mixture for 10 min. The
solution was then removed from the head
source and left at room temperature.
Following this step the extract was then
filtered through a Whatman filter paper No.1.
The extract was kept in refrigerator at 4ºC
for further experiments.
2.3 Qualitative Phytochemical Analysis:
Phytochemical components of the aqueous
extracts of betel nut and betel leaf were
screened by using standard methods. The
components analyzed were Alkaloids,
Flavonoids,
Anthroquinone,
Saponins,
Phenol, Protein, Coumarin, Reducing Sugar,
Tannins, Phytosterols, Anthrocyanides,
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2.3.9 Phytosterols: Four mg of the plant
sample was treated with 0.5 ml of acetic
anhydride and 0.5ml of chloroform. Then
concentrated sulphuric acid was added
slowly and green bluish color for steroids
was observed (Siddiqui and Ali, 1997).
2.3.10 Terpinoids (Keller-kilani test): To 4
mg of the sample was treated with 0.5 ml of
acetic anhydride and 0.5ml of chloroform.
Concentrated sulphuric acid was added
slowly along the sides of the test tube. The
presence of red violet colour was observed
for terpinoids.
2.3.11 Protein: To 1 ml of the extract few
drops of Barfoed’s reagent was added to give
blue color products to indicate the present of
protein.
2.3.12 Anthroquinone: To 0.5 g of the
sample was boiled with 10ml of dilute
sulphuric acid and filtered while hot. The
filtrate was shaken with 5 ml of chloroform.
The chloroform layer was pipette out in to
another test tube and one ml of dilute
ammonia was added. The resulting solution
was observed for color changes (Ayoola et
al., 2008).
2.3.13 Anthocyanins: (i) To the substance,
10% sodium hydroxide was added; blue
color shows the presence of anthocyanins.
(ii) To the substance conc. sulphuric acid
was added, yellowish orange color confirms
the presence of anthocyanins. (iii) 2 ml of
aqueous extract is added to 2 ml of 2N HCl
& NH3, the appearance of pink red turns blue
violet indicates presence of Anthocyanin
(Abollfazl et al., 2012).
2.3.14 Coumarins: To 1 ml of extract, 1 ml
of 10% sodium hydroxide was added. The
presence of coumarins is indicated by the
formation of yellow color (Abollfazl et al.,
2012).
2.3.15 Phlobatannins: Deposition of a red
precipitate when 2mls of extract was
boiled with
1ml
of 1% aqueous
hydrochloric acid was taken as evidence for
the presence of phlobatannins (Solomon et
al., 2013).
2.3.16 Acids: One ml of plant extract treated
with sodium bi carbonate results in
formation of effervescence presence of acid
(Raaman, 2006).
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3. RESULTS AND DISCUSSION
3.1 Phytochemical screening of aqueous
extract of betel nut
The preliminary photochemical screening
of the aqueous extraction of betel nut was
reported (Table 1).The positive result for the
presence of alkaloids, saponins, phenol,
reducing sugar, triterpinoids and glycosides
substance observed in aqueous extract of
betel nut. However, the negative results for
the absence of flavonoids, protein, coumarin,
tannins, phytosterols, anthroquinone, anthrocyanides, phlobatannins and acids substance
in aqueous extract of betel nut. Shivasankar
and Govindarajan (1963) reported that the
recant
contains
main
biochemical
compounds such as polyphenol (20%), fat
(15%), starch (20%) and alkaloids (0.5%).
Madhusudan et al. (2012) demonstrated the
preliminary phytochemical screening of
Areca catechu extract had showed that
amount of arecoline present in wet Areca
catechu was higher than that of dried Areca
catechu extract. Earlier study clearly
indicated areca seeds had high contents of
phenols and flavonoids and high related
antioxidant activity (Zhang et al., 2011).
Bravo (2008) study reported that the Areca
catechu plant contains a diverse group of
phenolic compounds with antioxidant
activity, as well as flavonoids, lignans,
stilbenes and simple phenolic acids,
including hydroxybenzoic acids
and
hydroxycinnamic acid. Existing studies of
areca nuts concern the properties of phenolic
compounds and alkaloids, pharmacology
activity (Zhang et al., 2008; Zhang et al.,
2008; Zhang et al., 2009). Similarly,
preliminary phytochemical studies of
aqueous extract of betel nut revealed the
physical characters of the extract. The
presence of active phytoconstituents such as
alkaloids, saponins, phenol, reducing sugar,
triterpinoids and glycosides were identified
by qualitative chemical tests (Fig. 1a).
3.2 Phytochemical screening of aqueous
extract of betel leaf
The preliminary photochemical screening
of the aqueous extraction of betel leaf was
reported (Table 1). The positive result for the
presence of alkaloids, saponins, coumarin,
and glycosides substance observed in
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J. Biosci. Nanosci., 3(1):2016
aqueous extract of betel leaf. However, the
negative results for the absence of
flavonoids, phenol, protein, reducing sugar,
tannins,
phytosterols,
anthroquinone,
anthrocyanides, triterpinoids, phlobatannins
and acids substance in aqueous extract of
betel leaf.
Table 2.1: Preliminary phytochemical analysis
Betel Nut
Betel Leaf
Aqueous
Aqueous
extract
extract
Alkaloids
+
+
Flavonoids
Saponins
+
+
Phenol
+
Protein
Coumarin
+
Reducing sugar
+
Tannins
Phytosterols
Anthroquinone
Anthrocyanides
Triterpinoids
+
Phlobatannins
Acids
Glycosides
+
+
(+ = Present and - = absence of phytoconstituents)
Phytochemicals
compound
Periyanayagam et al. (2012) study
revealed that the preliminary phytochemical
screening of Piper betle L. showed the
presence of flavonoids, terpenoids, steroids,
volatile oil, mucilage, tannin, saponins,
carbohydrates, reducing sugars, proteins and
amino acids and absence of alkaloids,
cyanogenetic glycosides, anthroquinone
glycosides, cardiac glycosides, fixed oils.
Sugumaran et al. (2011) investigation on
betel leaves revealed the presence of
alkaloids, carbohydrate, amino acids, tannins
and steroidal components. Previously many
researches works indicated the betel leaves
contains starch, diastases, sugars and an
essential oil composing of safrole, allyal
pyrocatechol
monoacetate,
eugenol,
terpinen-4-ol, eugenyl acetate, etc. as the
major components and the middle part of the
vine contains major superiority of tannin
(Chopra and Chopra, 1958; Kanjwani et al.,
2008). Hence, our present study revealed that
the aqueous extract of betel leave showed
presence of alkaloids, saponins, coumarin,
and glycosides substance (Fig. 1b).
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Fig. 1: Phytochemicals of aqueous extract of
a) betel nut and b) betel leaf
4. CONCLUSION
In the present work was undertaken with a
preliminary phytochemical screening of betel
nut and betel leaves showed the presence of
active compound in the aqueous extract. The
positive result for the presence of alkaloids,
saponins,
phenol,
reducing
sugar,
triterpinoids and glycosides substance
observed in aqueous extract of betel nut. The
aqueous extract of betel leaves showed the
presence of preliminary phytochemical such
as alkaloids, saponins, coumarin and
glycosides substance.
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