Estimation of flavonoid lantana camara linn verbenaceae

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_________________________________
* Corresponding author: Vedavathi.T
E-mail address: vedavathi.thava@yahoo.com
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Print ISSN: 2278 – 2648
Online ISSN: 2278 - 2656 IJRPP | Volume 2 | Issue 1 | 2013 Research article
Estimation of flavonoid, phenolic content and free radical scavenging activity
of fresh unripe fruits of lantana camara linn (verbenaceae)
*Vedavathi T, Bhargavi K, Swetha G, Mythri K.
CMR College of Pharmacy, Kandlakoya, Medchal Road, Hyderabad-501 401.
ABSTRACT
The Lantana camara Linn (verbenaceae) is available throughout world as a weed. The genus Lantana includes 2500
species worldwide and is known for its bioactive secondary metabolites and essential oils. It is used for the treatment
of various disorders. The therapeutic effects of tannins and flavonoids can be largely attributed to their free radical
scavenging property and it is responsible for many pharmacological activities. The present study is designed for the
extraction of fresh unripe fruits using alcohol (cold, hot and micro wave techniques). The extracts were investigated
for phytochemicals, total phenolic components, flavonoidal content and in-vitro free radical scavenging property
(DPPH–RSA method). The preliminary phytochemical investigations revealed that presence of glycosides,
alkaloids, carbohydrates, flavonoids, inuline, tannins (phenolic compounds). The total Phenolic content of alcoholic
extracts (cold, hot and micro wave) showed the content values of 4.67±0.22% w/w, 6.92±0.41% w/w and
7.291±0.13%w/w and total flavonoids estimation of alcoholic extracts (cold, hot and micro wave methods) showed
the content values of 5.12±0.08% w/w, 6.623±0.32%w/w and 7.458±0.24%w/w for Quercetin respectively. Further
investigation were carried out for In-vitro free radical scavenging assay by calculating its % inhibition by means of
IC50 values, all the extracts concentration has been adjusted to come under the linearity range and here many
reference standards like Tannic acid, Quercetin, Ascorbic acid have been taken for the method suitability. The
results revealed that fresh unripe fruits of this plant have free radical scavenging potential. Among these results
alcoholic extract by micro wave technique has more yield and more potent than other extraction methods. In
conclusion that the Lantana camara Linn. (Verbenaceae) fresh unripe fruits possesses the antioxidant substance
which may be potential responsible for the treatment of tumors and rheumatism and other oxidative stress related
diseases.
Keywords: Lantana camara Linn. (Verbenaceae), Total phenolic content (TPC), Total flavonoid content (TFC),
radical scavenging assay (DPPH – RSA)
INTRODUCTION
The plant Lantana camara Linn, family
Verbenaceae is also known as wild sage, red sage,
Spanish flag and is also available throughout central
and south India in most dry stony hills and black
soil[1]
. Fruits are small, 5 mm diameter, greenish-
International Journal of Research in
Pharmacology & Pharmacotherapeutics

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blue, blackish, drupaceous, shinning with two nut lets
almost throughout the year and dispersed by birds.
Seeds germinate very easily. The chemical
constituents of lantana camara are caryophyllene, 1-
α-phellandrene, lantadene-A, lantadene-B[1]
,
flavoniods[2]
, saponins, tannins, γ- gurjunene[3]
,
lancamarone quinine, lantanine etc.
The plant is vulnerary, diaphoretic, carminative, antis
pasmodic and tonic. In many parts of the world the
plant has been used to treat a wide variety of
disorders like anti-inflammatory[4]
, anti-malarial[5]
,
fungicidal[6]
, eczema[7]
, rheumatism[8]
, asthma[9]
,
inhibitor of acetyl cholinesterase[10]
, abortificient[11]
,
skin diseases[12]
, high blood pressure and biological
control[13]
. In the folk medicine, it is used especially
for tumors and cancer [1]
. A tea prepared from the
leaves and flowers is taken against fever, influenza
and stomach ache. In Central and South America the
leaves were made into a poultice to treat sores,
chicken pox and measles. In Ghana infusions of the
whole plant is used against bronchitis. The powdered
root in milk was given to children for stomach ache.
In Asian countries leaves are used for cuts,
rheumatism, ulcers and as a vermifuge. Decoctions
are applied externally against leprosy and scabies [14]
.
In India the leaves of the plant are boiled for tea and
the decoction is a remedy against cough. The
decoction of the whole plant is given as treatment
against tetanus, rheumatism, malaria and ataxia of
abdominal viscera. It is used as a lotion for wounds.
Leaf powder is applied to cuts, ulcers and swellings
[15]
.
There are some of the reports related to antioxidant
activity, total phenolic, total flavonoidal content
estimations on total plant, leaves, ripe fruits etc. But,
there is no proper scientific studies have been
reported for the fresh unripe fruits of this plant. Due
to this considerable savings in the time, energy and
active constituents (degradation is also less), the
novel microwave method of extraction is used to
compare with the traditional methods [16]
. So the aim
of the study is to compare the yield value of the
extract and to investigate the total phenolic content,
total flavonoidal content and in-vitro free radical
scavenging activity of alcoholic extracts (cold, hot
and micro wave techniques) of fresh unripe fruits of
this plant.
An antioxidant is a molecule capable of slowing or
preventing the oxidation of other molecules.
Oxidation is a chemical reaction that transfers
electrons from a substance to an oxidizing agent.
Oxidation reactions can produce free radicals, which
start chain reactions that damage cells. Antioxidants
terminate these chain reactions by removing free
radical intermediates and inhibit other oxidation
reactions by being oxidized themselves. As a result,
antioxidants are often reducing agents such as
ascorbic acid or polyphenols [17]
. However, it is
unknown whether oxidative stress is the cause or the
consequence of disease. Antioxidants are also widely
used as ingredients in dietary supplements in the
hope of maintaining health and preventing diseases
such as cancer and tumors. Although initial studies
suggested that antioxidant supplements might
promote health and suggested instead that excess
supplementation may be harmful. In addition to these
uses of natural antioxidants in medicine, these
compounds have many industrial uses, such as
preservatives in food and cosmetics[18]
.So that we got
an interest to study by means of preliminary in-vitro
antioxidant work which we have carried out in fresh
unripe fruits of Lantana camara.
MATERIAL AND METHODS
Chemicals and reagents
Chemicals used in this study were 1, 1-diphenyl-2-
picryl hydrazyl (DPPH) obtained from John Baker
Inc Colorado, U.S.A, Quercitin, tannic acid, ascorbic
acid, aluminium chloride, sodium hydroxide, folin-
denis reagent (Phosphomolybdic acid, sodium
tungustate, phosphoric acid), sodium carbonate and
sodium nitrite are obtained from SD Fine Chemicals
Ltd, India. All other reagents and solvents used in the
study are of analytical grade.
Plant material
Fresh unripe fruits of Lantana camara Linn family
Verbenaceae were collected from surrounding CMR
College of Pharmacy, Medchal, Rangareddy district,
India during the month of June. The fresh unripe
fruits were crushed for extraction purpose.
Preparation of crude extracts
Alcoholic extract
100 g of fresh unripe fruits of Lantana camara Linn
(Verbenaceae) were weighed and crushed in an
electronic mixer and were soaked in 200 ml of
alcohol for about 2 days in 500ml borosilicate glass

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jars for each method (cold, hot and microwave) of
extraction processes.
Cold extraction
The imbibed material after two days was filtered
through cotton bag and squeezed.
Hot extraction
The imbibed material was packed in Soxhlet
apparatus with the help of cotton bag and extracted at
50 - 55ºc and continued until the complete exhausting
of crude drug (no color in the siphon tube). The
cotton bag was removed from extractor, pressed the
marc and added the contents to RBF to distill the
solvent [19]
.
Micro wave extraction
The imbibed material was taken as suspension (50ml)
in solvent in a pyrex cylindrical tubes. Tubes are
irradiated in a Microwave oven (450W) and the
extracts were not allowed to boil, therefore, after 30 s
they were cooled to room temperature for few min;
the irradiation step was repeated for 20 times in the
same way, it gives corresponding extract [16]
. After
cooling, it is filtered through a cotton bag.
To prepare dry powder form of extract
The dry powder of this extract was prepared by
concentrating the resultant filtrates by distillation of
the solvent and the resultant concentrate was dried by
using the simple saloon water sprayer, by spraying
the extract on the glass plates, after the predetermined
flow conditioned consistency thick solution was
poured into the sprayer (here the above concentrated
extract solution varies to nature of plant material), by
which it was air dried. The clumpy dry powder
obtained was scraped by the knife from the plate and
packed in air tight plastic container and stored in
vacuum desiccators as such. The preconditioned set
method can be optimized by evaluating the
quantitative test of any existed constituents like
phenols, flavonoids by suitable validated methods.
This present study was undertaken by the
spectrophotometric method.
Yields of various extracts
Cold extraction process – 3.46 Gms
Continuous hot extraction by Soxhlet apparatus -
4.92 Gms
Microwave process – 6.12 Gms
Qualitative Preliminary Phytochemical
screening
The extracts thus obtained were subjected to
preliminary phytochemical screening [20]
. The results
obtained in the present investigation of fresh unripe
fruits of Lantana camara Linn showed in the table 1.
TABLE-1: Phytochemical analysis of various extracts of fresh unripe fruits of Lantana
camara Linn.
Test of extract Hot extract Cold extract Microwave Extract
Alkaloids
Aminoacids
Carbohydrates
Flavanoids
Glycosides
Inulin
Tannins
Proteins
Starch
Napthoquinones
+
-
+
+
-
+
+
-
-
-
+
-
+
+
+
+
+
-
-
-
+
-
+
+
-
+
+
-
-
-
Note: ‘+’ Positive, ‘-’ Negative

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Estimation of TPC by UV – Visible
spectrophotometer
By Folin – Denis Method
This method is based on the oxidation of molecule
containing a –OH groups. The tannin and tannin like
compound reduce Phosphotungustomolybdic acid in
alkaline solution to produce a highly blue colored
solution [21, 22]
. 100 mg of the each powder of extract
was dissolved and diluted with distilled water and
that has adjusted to come under the linearity range
i.e. (50 μg/ml) of the drug was withdrawn in 10ml
volumetric flask separately. To each flask 0.5 ml of
Folin-Denis reagent and 1ml of Sodium carbonate
was added and volume is made up to 10 ml with
distilled water. The absorbance was measured at
absorption maxima 700 nm within 30 minute of
reaction against the blank. The total phenolic content
was determined by using calibration curve (5 to 25
μg/ml). Three readings were taken for each and every
solution for checking the reproducibility and to get
accurate result. Results are provided in (Table-2 and
Figure-1). The intensity of the solution is
proportional to the amount of tannins and can be
estimated against standard tannic acid, the total
phenolic content, expressed as mg tannic acid
equivalents per 100g dry weight of sample
.
FIGURE 1: Results of Total Phenolic content
R2
values represented mean data set of n=3
Table 2: Results of Total phenolic content
No Concentration of extract % w/w of total tannin
1 Alcoholic cold 4.67± 0.22
2 Alcoholic hot 6.92± 0.41
3 Alcoholic microwave 7.291±0.13
Values are mean ± S.E.M, n=3
Total Flavonoid Content by
Spectrophotometer Aluminum chloride
visible Spectrophotometric assay method
Total flavonoid contents were measured with the
aluminum chloride visible Spectrophotometric assay
[23]
. Alcoholic extracts that has been adjusted to come
under the linearity range i.e. (400 μg/ml) and
different dilution of standard solution of Quercetin
(10-50 μg/ml) were added to 10ml volumetric flask
containing 4ml of water. To the above mixture, 0.3ml
of 5% NaNO2 was added. After 5 minutes, 0.3ml of
10% AlCl3 was added. After 6 min, 2ml of 1 M
NaOH was added and the total volume was made up
to 10ml with distill water. Then the solution was
mixed well and the absorbance was measured against
y = 0.006x + 0.001
R² = 0.998
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
0.16
0.18
0 5 10 15 20 25 30
Standard Graph for Tannic acid
(TPC)

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a freshly prepared reagent blank at 510 nm. Results
are provided in (Table 3 and Figure 2) Total
flavonoid content of the extracts was expressed as
percentage of Quercetin equivalent per 100 g dry
weight of sample.
FIGURE 2: Results of Flavonoid content
R2
values represented mean data set of n=3
Table 3: Results of Flavonoids content
No Concentration of extract %w/w of total flavonoid
1 Alcoholic cold 5.12 ± 0.08
2 Alcoholic hot 6.623± 0.32
3 Alcoholic microwave 7.458±0.24
Values are mean ± S.E.M, n=3
DPPH –RSA method
The free radical scavenging activity of each of
alcoholic extracts and the standard L-Ascorbic Acid
(Vitamin C) was measured in terms of hydrogen
donating or radical scavenging ability using the stable
radical DPPH[24,25]
. Here, 0.1mM solution of DPPH
in alcohol was prepared and it must be protected from
light influence by maintaining the dark condition and
also fold by aluminum foil and 3 ml of this solution
was added to 1ml various conc. (10-50 µg/ml) of
extracts or standard solution of (10-50 µg/ml).
Absorbance was taken after 30 min at 517nm.
Results are provided in (Table 4 and Figure 3-6).
% inhibition activity = A0 – A1 X 100
A0
Where, A0 is the absorbance of the control,
A1 is the absorbance of extract/standard taken as
Ascorbic acid.
y = 0.001x + 0.001
R² = 0.998
0
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
0.09
0.1
0 10 20 30 40 50 60
Standard Graph for Quercetin

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FIGURE 3: Results of DPPH radical scavenging assay
R2
values represented mean data set of n=3
FIGURE 4: Results of DPPH radical scavenging assay
R2
values represented mean data set of n=3
FIGURE 5: Results of DPPH radical scavenging assay
R2
values represented mean data set of n=3
y = 1.649x + 1.318
R² = 0.998
0
20
40
60
80
100
0 10 20 30 40 50 60
STD Ascorbic Acid
y = 1.643x + 0.669
R² = 0.996
0
20
40
60
80
100
0 10 20 30 40 50 60
Alcoholic Cold Extract
y = 1.492x + 0.511
R² = 0.998
0
10
20
30
40
50
60
70
80
0 10 20 30 40 50 60
Alcoholic Hot Extract

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FIGURE 6: Results of DPPH radical scavenging assay
R2
values represented mean data set of n=3
RESULTS
Effect of TPC & Flavonoid content
The quantitative determination of the total phenolic
content, expressed as mg tannic acid equivalents and
per 100g weight of sample. TPC of alcoholic extracts
(cold, hot and microwave) showed the content values
of 4.67µg/ml, 6.92µg/ml and 7.29µg/ml and total
flavonoid content of the extracts was expressed as
percentage of Quercetin equivalent per 100 g dry
weight of sample. TFC estimation of alcoholic
extracts (cold, hot and microwave) showed the
content values of 5 µg/ml, 6.625µg/ml and 7.5µg/ml
The above results showed that alcoholic microwave
extraction contain more tannins and flavonoid
content than the alcoholic hot and cold extract.
Capacity of DPPH –RSA
The antioxidant reacts with stable free radical, DPPH
and converts it to 1, 1- Diphenyl-2- Picryl Hydrazine.
The ability to scavenge the free radical, DPPH was
measured at an absorbance of 517 nm. So the DPPH
– RSA and its %inhibition of alcoholic extracts (cold,
hot and microwave) showed that IC50 values
27.81μg/ml (R2
=0.996), 33.76 μg/ml (R2
=0.997) and
36.87μg/ml (R2
=0.998) respectively. Ascorbic acid
has taken as reference which showed 37.96μg/ml.
(R2
=0.998) among these results microwave extract
has more potent than hot and cold extracts. The
overall results of % inhibition as shown in the (Table
4) respective to IC50 values and regression R2
is the
mean value of (n=3).
Table 4: % Inhibition by DPPH-RSA
Samples Equation * R2
values IC 50 values
Standard Ascorbic Acid y = 1.649x + 1.318 0.998 37.96μg/ml
Alcoholic Cold Extract y = 1.643x + 0.669 0.996 27.81μg/ml
Alcoholic Hot Extract y = 1.492x + 0.511 0.998 33.76μg/ml
Alcoholic Microwave Extract y = 1.627x + 1.300 0.998 36.87.μg/ml
*Data set of n=3 and mean R2
values obtained from the graphs
DISCUSSION
The fresh unripe fruits Lantana camara Linn family
Verbenaceae possesses the antioxidant property
substance which may be potential responsible for the
treatment of cancer and tumors there is much scope
for fresh unripe fruit portion and more number of
studies can be undertaken like oxidative stress,
y = 1.627x + 1.300
R² = 0.998
0
10
20
30
40
50
60
70
80
90
0 10 20 30 40 50 60
Alcoholic Microwave Extract

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hepatoprotective, nephroprotective, anticancer, skin
disorders, rheumatism, asthma etc. In future we look
forward to check the potency of the fruits by means
of in-vivo anti-oxidant studies.
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