Department of Botany & Microbiology, Acharya Nagarjuna University,
Guntur, Andhra Pradesh, India.
Since ancient times, about 80 % of individuals
compounds derived from medicinal plants
Several hundred plant species and herbs in the
purified constituents are used in indigenous
system of medicines and are of great
importance to the health of individual and
communities, which have ultimately evaluated
into the modern therapeutic science. Medicinal
plants are important source of life saving drugs
for majority of the world population
Phytochemical constituents are non-nutritive plant chemicals that have preventive and curative
properties of disease. The use of plants and phytochemicals, both with known biological properties,
can be of great significance in therapeutic treatment. The present study includes phytochemical
screening and quantification of secondary metabolites and their biological activities of root extract
of Syzygium samarangense. Phytochemical screening of the plant root extracts with ethyl acetate,
methanol and water showed the presence of flavonoids, terpenoids and phenolic compounds. In
aqueous extracts, Terpenoids with highest quantity (81.923 micrograms per gram extract) were
estimated, whereas flavonoids are present only in methanolic extract and with an estimated quantity
of 33.687 µg /per gram extract. Anti oxidant, anti inflammatory, and anti diabetic activity of these
root extracts reveal that methanoilc extracts with flavonoids showed the high antioxidant, anti
inflammatory and antimicrobial activity than the aqueous and ethyl acetate extract. Highest
antidiabetic activity was observed in aqueous extract. Antimicrobial activity study reveals that gram
positive bacterial strains are more sensitive than the gram negative bacteria to all the three types of
root extracts. The present study reveals that the root extract of Syzygium samarangense is a potential
source for phytochemicals for traditional use as therapeutics.
Fig. 1: Syzygium samarangense (wax jambu) plant and fruit
Syzygium samarangense (common name - wax
jambu) is a plant species in the family Myrtaceae
which is widely cultivated in the tropics
leaves, root, bark, fruits of the plant
Activity, Anti hyperglycaemic activity, Analgesic
and Anti- Inflammatory activity are reported with
various parts of the plant
. And also
constituent to be tannins, Quercetin glycosides,
monoterpenes secondary metabolites those
involved in pharmacological properties
Traditionally the root bark decoction of the
and amenorrhea and also used as abortifacient.
Root is used as diuretic and is given to alleviate
edema. Malayans use powdered dried root
preparations for itching. As plant root has
significant therapeutic uses, this study is aimed
to screen the phytochemicals of the root and
study of its biological activities.
The literature review proves that the plant is rich
with many medicinal and bioactive compounds
like flavanoids, phenolic compounds, glycosides,
terpinoids etc. various studies are reported
these compounds in various areal parts of the
plant. K. M. Moneruzzaman et al
, reported the
(326.67 mg GAE/100g) content from the
Syzygium samarangense plant where in our
study root is reported as 3mg/100grams. Wu
constituents from the branches and leaves with
95% ethanol, then partitioned with petroleum
ether, chloroform and ethyl acetate. M.O.
amounts of saponins content (4.77%). Mario J.
, reported the six quercetin
, reported the total phenolic content
(310Î¼g/mg) of Syzygium samarangense Fruit
extract (SSFE) where the study with roots are
reported 46.944 µg and 23.056 µg per gram in
methanolic and water extract respectively. G. R
Nair et al
, reported the two flavonol
flavonoids isolated from the hexane extract.
, isolated four rare
flavanone the compounds from the hexane
extract of the leaves. Dennis D. Raga et al
stearate (1b), sitosteryl stearate (1c), and 24-
methylenecycloartanyl stearate (1d) (sample 1)
from the air-dried leaves. Samy MN et al
isolated three new compounds (one new
the leaves. Rachana Srivastava et al
native form and 4′,6′dihydroxy2′ methoxy3′,
5′dimethyl chalcone along with ursolic acid,
jacoumaric acid and arjunolic acid have been
isolated from the aerial parts. However the study
with root extracts reported that root reported with
high terpenoid content which is not reported
earlier. And it is also reported that root is
another alternate source for flavonoid and
phenoilc groups. Anti oxidant, anti inflammatory,
and anti diabetic activity of these three root
extracts conducted in order to study the
biological activity of the plant. Anti oxidant
activity of the extract was conducted by DPPH
methanolic extracts which consist of flavonoids
has shown the high antioxidant activity
(88.021%) (table-3 and graph A) than the
aqueous and ethyl acetate extracts at 40 µg/ml
extract concentration. Similar studies are
reported by the Fonseca A et al
of the fruit. Vasanthi et al
, reported that IC50
found to be 140 Î¼g/ml in standard (Lascorbic
acid), 280 Î¼g/ml in SSFE by DPPH and
ABTS.+ scavenging activity found to be the IC50
value 175 Î¼g/ml and 250 Î¼g/ml respectively.
No one has reported with root extracts and
found that similar antioxidant activity as fruit.
Antidiabetic activity (table-4 and graph B) of the
root extracts was studied by In vitro
Results showed that dose dependent inhibition
was observed with all the samples where as
aqueous extract (92.626%) exhibited the highest
activity than remaining two extracts. No
antidiabetic activity studies are reported with S.
5) of the root also studied with all three extracts
and among them methanolic extracts (84.552%)
showed the highest anti inflammatory activity. In
literature Shabnam Mollika et al
activity of the methanolic extract of leaves in
mice. Dennis D. Raga et al
, reported that the
inflammatory activities at effective doses of 6.25
mg/kg body weight and 12.5 mg/kg body weight,
respectively. This reveals theat no one has
reported the anti inflammatory activity with root
and potential activity was reported in our study.
Antimicrobial activity result (table-6 and figure-3)
reveals that Gram-positive bacteria were more
sensitive than Gram-negative ones towards the
plant extracts studied. Bacillus subtilis and
inhibition zone than gram negative Escherichia
antimicrobial activity of the different areal plant
oil from the leaf of Syzygium samarangense on
the leaves of minimum inhibitory concentration
(MIC) value was determined to be 20 mg/mL. K.
Venkata Ratnam et al
, evaluated the
bacterial and fungal strains with petroleum ether
and methanol extracts found to be effective on
both Gram positive and Gram negative bacteria.
, reported that juice extracts
against Escherichia coli, Salmonella typhi and
, elucidated the dichloromethane extract of
against C. albicans, low activity against T.
mentagrophytes and low antibacterial activity
against E. coli, S. aureus and P. aeruginosa. It
was inactive against B. subtilis and A. niger. In
comparison with the literature studies root
extracts of S. samarangense also have
significant antimicrobial active compounds with
low minimum inhibition concentration. Studies
proved that compounds in plant extracts have
potential activity against gram positive bacteria
than gram negative.
MATERIAL AND METHODS
Chemicals: The solvents used for root
extraction are Methanol and Ethyl Acetate. The
reagents used for phytochemical screening and
estimation were of laboratory reagent grade and
were purchased for Merck chemicals private
limited, Mumbai, Fisher scientific, Mumbai and
SD fine chemicals Mumbai. Distilled water has
been used for aqueous extraction. Alpha
amylase enzyme was purchased from Sigma
Aldrich chemicals, Ofloxacin drug.
UV 2301 Double Bean UV
apparatus, heating mantle, incubator, autoclave.
Sample collection: Root material of Syzygium
in various places of East Godavari district,
Andhra Pradesh, India. The roots are separated
and allowed to shade dry. The root sample was
ground and powdered for solvent extraction.
present in the roots of the collected plants were
isolated using different solvents like ethyl
acetate, methanol and water in a series of
extraction method from low polarity to high
polarity using soxhlet extraction method.
The bacterial strains used for screening of
antimicrobial activity are Salmonella typhi,
Escherichia coli, Pseudomonas aeruginosa,
Phytochemical analysis of the root extracts was
performed by following standard methods
Few drops of concentrated sulphuric acid are
added to the plant extract, shaken and on
standing; lower layer turns red in colour.
To the extract, few drops of acetic anhydride
was added and mixed well. 1 ml of
concentrated sulphuric acid is added from the
sides of test tube, a reddish brown ring is
formed at the junction of two layers.
2. Tests for triterpenoids
added to the extract, shaken and on
standing, lower part turns golden yellow
Lieberman Burchard’s Test
sides of test tube, a red ring indicates
Excess of acetyl chloride and pinch of zinc
chloride are added to the extract solution,
kept aside for reaction to subside and
warmed on water bath, cosin red colour is
To the extract, few drops of chlorosulfonic
acid in glacial acetic acid (7:3) are added, red
colour is produced.
3. Test for Saponins
Small amount of extract is shaken with little
quantity of water; the foam produced persists
for 10 minutes. It confirms the presence of
To 2ml of 1.8% Sodium chloride solution in
two test tubes, 2ml distilled water is added to
one and 2ml of 1% extract to the other, 5
drops of blood is added to each tube and
gently mixed with the contents. Haemolysis
observed under the microscope in the tube
containing the extract indicates the presence
The extract is hydrolysed with sulphuric acid and
extracted with chloroform. The chloroform layer
is tested for steroids.
5. Tests for Triterpenoidal Saponin
is tested for triterpenoids.
6. Tests for Alkaloids
The acid layer when mixed with Mayer’s
reagent (Potassium mercuric iodide solution)
The acid layer with a few drops of
reagent (Potassium bismuth
iodide) gives reddish brown precipitate.
The acid layer when mixed with few drops of
Wagner’s reagent (solution of iodide in
potassium iodide) gives brown to red
Hager’s reagent (Saturated solution of pricric
acid) gives yellow coloured precipitate.
The extract when heated with Fehling’s A
and B solutions gives an orange red
precipitate showing the presence of reducing
The extract is treated with Molisch’s reagent
and concentrated sulphuric acid along the
shows the presence of carbohydrate.
The extract on heating with
reagent, brown precipitate indicates the
presence of sugar.
Barfoed’s reagent is added and boiled on
8. Test for Flavonoids
The extract solution with a few fragments of
hydrochloric acid produced magenta colour
after few minutes.
Alcoholic solution of extract reacts with
freshly prepared ferric chloride solution and
given blackfish green color.
Alcoholic solution of extract reacts with 10%
lead acetate solution and given yellow
Drug is powdered and extracted with either
ammonia or caustic soda. The aqueous layer
shows pink color
This is for cardiac glycosides. The extract
and 0.4 glacial acetic acid are mixed with
ferrous chloride and 0.5 mi of concentrated
sulphuric acid. The acetic acid layer shows
10. Test for Phenolic Compounds
Ferric chloride test
Treat the extract with ferric chloride solution
then blue color appears if hydrolysable
tannins are present and green color appears
if condensed tannins are present.
To the test solution add 1% gelatin solution
containing 10% NaCl, and then precipitate is
Treat the test solution with aqueous ammonia
and expose to air gradually, green color is
The total phenolic content in different solvent
extracts was determined with the Folin-
Ciocalteu’s reagent (FCR). In the procedure, 1ml
of extract was mixed with 0.4 ml FCR (diluted
1:10 v/v). After 5 min 4 ml of sodium carbonate
solution was added. The final volume of the
tubes were made up to 10 ml with distilled water
and allowed to stand for 90 min at room
temperature. Absorbance of sample was
measured against the blank at 765nm using a
spectrophotometer. A calibration curve was
constructed using gallic acid solution as
standard and total phenolic content of the
extract was expressed in terms of milligrams of
gallic acid per gram of dry weight.
Total flavonoid content was determined using
aluminium chloride (AlCl
) according to a known
extract (1 ml) was added to 3 ml distilled water
followed by 5% NaNO
(0. 3ml). After 5 min at
(0.3 ml, 10%) was added. After
with 2.0 ml of 1 M NaOH. Finally, the reaction
mixture was diluted to 10ml with water and the
absorbance was measured at 510 nm. A
quercetin solutions as standard and total
terms of milligrams of quercetin per gram of dry
Quantitative estimation of terpenoids
To 1ml of plant extract 2 ml of chloroform was
mixed in extract of plant sample and 3 ml of
sulphuric acid were added in sample extract.
Reddish brown color was obtained in the test
tube. Final volume in the test tube was made up
to 10ml with water. The absorbance of the
formed color was measured at 538nm against
reagent blank prepared similarly without plant
Measurement of Antioxidant Activity using
The antioxidant activity of the different root
extracts was determined on the basis of their
scavenging activity of the stable 1, 1- dipheny1-
2-picryl hydrazyl (DPPH) free radical. DPPH is a
stable free radical containing an odd electron in
its structure and usually utilized for detection of
the radical scavenging activity in chemical
analysis. lml of each solution of different
concentrations (1-500g/m1) of the extracts was
added to 3 ml of 0.004% ethanolic DPPH free
absorbance of the preparations were taken at
517 nm by a UV spectrophotometer which was
compared with the corresponding absorbance of
standard ascorbic acid concentrations (1-
500µg/m1).The method described by Hatano et
some modifications. Then the % inhibition was
calculated by the following equation:
1ml of alpha amylase and 1 ml of plant extract in
a test tube and incubated at 37
C for 10 min.
solution was added to each tube and incubated
at 37◦C for 15min. The reaction was terminated
with 2 mL DNSA reagent, placed in boiling water
bath for 5min, cooled to room temperature,
diluted, and the absorbance measured at 546
nm. The control reaction representing 100%
enzyme activity did not contain any plant extract.
To eliminate the absorbance produced by plant
extract, appropriate extract controls were also
A solution of 0.2% W/V of BSA was prepared in
Tris buffer saline and PH was adjusted to 6.8
using glacial acetic acid. Stock solutions of plant
extract were prepared by using methanol as a
solvent. From these stock solutions 6 different
concentrations of 10-500µg/ml were prepared by
using methanol as a solvent. 50µl of each
extract was transferred to Eppendorf tubes using
1ml micro pipette. 5ml of 0.2% W/V BSA was
added to all the above Eppendorf tubes. The
control consists of 5ml 0.2% W/V BSA solution
with 50 µl methanol. The test tubes were heated
at 72° C for 5 minutes and then cooled for 10
minutes. The absorbance of these solutions was
determined by using UV/Vis Double beam
wavelength of 660nm. The % inhibition of
precipitation (denaturation of the protein) was
determined on a % basis relative to the control
using the following formula.
Root extracts of Syzygium samarangense were
tested by agar well-diffusion method to
determine the antimicrobial activity. Nutrient
agar (NA) plates were seed inoculated. Wells
(10mm diameter and about 2 cm a part) were
made in each of these plates using sterile cork
borer. Stock solution of each plant extract was
prepared at a different concentrations 1000,
500, 200, 100, 50,10µg/ ml in different plant
extracts viz. Methanol, ethyl acetate, water.
About 100 µl of different concentrations of plant
solvent extracts were added with sterile syringe
into the wells and allowed to diffuse at room
temperature for 2hrs. Control experiments
comprising inoculum without plant extract were
set up. The plates were incubated at 37°C for
18-24 h. The diameter of the inhibition zone
(mm) was measured and the activity index was
also calculated. Triplicates were maintained and
the experiment was repeated thrice, for each
replicates the readings were taken in three
different fixed directions and the average values
were recorded. Oflaxacin drug was used as
standard antibacterial agent and compared with
the standard results.
Secondary metabolites present in the plants are
responsible for the biological activities such as
antimicrobial, antiinflammatory, anticarcinogenic,
antimalarial, anticholinergic, antileprosy etc. The
preliminary screening of phytochemicals and
evaluation of bioactive may lead to medicinal
plant drug discovery and development of
phytomedicine. In the present study the root of
systems. Among the three, aqueous extracts
were proved to contain more number of
compounds than other two solvents extracts. In
saponins, tannins, roteins and aminoacids,
terpenoids, phenolic compounds were identified.
Compounds were indentified in the methanolic
extract. In Ethyl acetate extract only Terpenoids
are identified. In continuation quantitative
analysis of the extracts has conducted to
estimate terpenoids, phenolic compounds and
flavonoids (table 1 and fig 2).
Terpenoids were detected with highest quantity
aqueous extracts than other phytochemicals.
Terpenoids were detected with 48.461µg
quantity in methanolic extract and 35.385 µg in
ethyl acetate extract. Whereas flavonoids are
only present in methanolic extract and amount of
flavanoid quantified was 33.687 µg /per gram
extract. Similarly phenolic compounds were
reported in methanolic and water extracts with
estimates as 30.156µg/g and 23.056 µg per
gram extract respectively. Our results were in
agreement with findings of the medicinal value
of plants lies in some chemical substances that
have definite physiological functions in the
human body. Different phytochemicals have
been found to possess a wide range of
medicinal properties, which may help in
protection against various diseases.
In the present investigation, primary and
secondary metabolites of the root were
qualitatively and quantitatively analyzed then the
inflammatory, and anti diabetic) was studied.
Further evaluation of phytochemicals and their
activity is needed for knowing the nutritional
potential as well as helpful in manufacturing new
The author is thankful to Head, Dept. of Botany
& Microbiology, ANU for providing the facilities
and also thankful to management, Hindu
permitting to do research analysis.
Proteins & Aminoacids
Table 2: Quantitative analysis of root extracts of S. samarangense
Amount found µg / g of extract
Table 3: Antioxidant activity (% DPPH scavenging activity) of S. samarangense root extracts
Graph B: Comparative graph of α
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