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COMPARATIVE EFFICACY OF PHYTOCHEMICAL ANALYSIS AND

ANTIOXIDANT ACTIVITY OF METHANOLIC EXTRACT OF
CALOTROPIS GIGANTEA AND CALOTROPIS PROCERA
*Hitesh Vashrambhai Patel, Jatin D. Patel, Bhautik Patel
Department of Biochemistry, Shree Alpesh N. Patel PG Institute, Charotar Education Society, Anand - 388 001, Gujarat, India.

ABSTRACT
Calotropis species is a common wasteland weed and are widely used as alternative therapeutic tool for the prevention
or treatment of many diseases. This study was designed to evaluate comparative antioxidant activity, metal chelating properties
and larvicidal activity of methanolic extract of two common species of Calotropis, viz. Calotropis gigantea (Linn.) R.Br. and
Calotropis procera (Ait.) R.Br. The total phenolic and flavonoid content was determined and expressed in term of gallic acid
equivalent and quercetin equivalent respectively. In this study, antioxidant activity was measured by radical (DPPH •)
scavenging, reducing power, FRAP assay and metal chelating activity assay. The leaves of C. procera were found to have
higher antioxidant potential than flower and root extract with IC50 values of 0.21μg/ml for DPPH scavenging, 0.98 mg/ ml
for metal chelating. C. procera significantly more potent in scavenging free radical and antioxidant activity than C. gigantea.
Extract of all parts of both species however demonstrated similar IC50 value for metal chelating activity. C. procera possess
comparatively higher antioxidant activity in reducing ferric ions than C. gigantea. The leaf methanolic extract showed a
concentration dependent larvicidal activity with a lowest LD50 value of 387 mg/l compared to other extracts. There was no
significant difference observed in the LC50 value for larvicidal activity of all parts between C. procera and C. gigantea which
indicate both species exhibits same effect against Ae. aegypti larvae. Occurrence of more total phenols and flavonoids in all
parts of C. procera as compared to C. gigantea correlates to its high antioxidant activity. The observations reported in this
paper could be of applied value in utilization of C. procera for different clinical purpose which showed strong antioxidant
potential rather than C. gigantea.

Key Words: Calotropis, Antioxidant activity, Larvicidal activity.

INTRODUCTION activities, including inhibition of ROS generation, direct or

Herbal medicine is becoming popular all over the indirect scavenging of free radicals and alteration of
world than the Allopathic medicine for medication. Much intracellular redox potential. Several medicinal plants have
attention has been focused on assessment of antioxidant been screened based on the integrative approach on drug
properties of various plants for finding new sources for development from Ayurveda (Mukherjee and Wahile,
natural antioxidants, functional foods and nutraceuticals. 2006). The traditional and folk medicinal system uses the
Natural antioxidants exhibits a wide range of biochemical plant products for the treatment of various infectious
diseases. The phytochemicals have been found to act as
Corresponding Author antioxidants by scavenging free radicals and may have
therapeutic potential for free radical associated disorders
Hitesh Vashrambhai Patel (Hausladen and Stamer, 1999).
Email: [email protected] There are two common species of Calotropis, viz.
Calotropis gigantea (Linn.) R.Br. and Calotropis procera
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(Ait.) R.Br. belongs to the family Asclepiadaceae (Singh, Dave, Botanist, M. B. Patel Science collage, Anand during
et al., 1990) which are known for its pharmacological December 2012. The plant parts were washed properly and
importance for centuries locally known as “Raktha Arka” dried in shade. Dried plant material was subjected to
and “Sweta Arka” respectively in India. It cultivated reduction to coarse powder using mechanical grinder,
throughout India in warm dry places from Punjab to passing through sieve #40 and stored in a tight container
western, central and southern India and is a soft-wooded,
evergreen, perennial shrub. The plant is reported to have Preparation of plant extract
diverse pharmacological actions (Sharma et al., 2011). The dried coarse powder material (100 g) was
Various parts of this plant such as leaves, stem, subjected to Soxhlet’s extraction separately and
flowers, and root bark are widely used in the folk medicine successively with methanol. The solvent was distilled
for the treatment of common disease such as fevers, under reduced pressure, controlled temperature (40-50°c)
rheumatism, indigestion, cough, cold, eczema, asthma, and the resulting semisolid mass was vacuum dried using
nausea, vomiting, and diarrhoea. It was reported that C. rotary flash evaporator to yield a solid residue put in
procera used in traditional medicine as a purgative, airtight container and stored in refrigerator. The following
anthelmintic, anticoagulant, anticancer as well as abbreviation has been used for each extract: C. gigantea:
antipyretic, analgesic (Sharma et al., 2012; Basu & Leaves (CGL), Flower (CGF) and root (CGR) and C.
Chaudhury, 1991). The plant produces milky white latex procera: Leaves (CPL), Flower (CPF) and root (CPR).
that exhibits analgesic activity and wound healing
properties in various animal models (Choedon et al., Phyhytochemical analysis
2006). The leaves are useful in the treatment of paralysis, Various phytochemical tests were carried out on
arthralegia, swelling and intermittent fevers. Methanolic the methanolic extract of all parts of both species of
and aqueous extracts of leaves of C. procera Linn, were Calotropis using standard procedures to identify the
subjected to the potential antioxidant and antibacterial phytoconstituents.
activity (Kareem et al. 2008). Root bark of C. gigantea
exhibits hepatoprotective activity, anticancer activity, Total phenolics and flavonoids content
antifertility, and anti-inflammatory activity. Cardiac An amount of total phenolics content in the
glycosides from C. gigantea exhibit anticancer properties extract were determined using a series of gallic acid as
(Bhat & Sharma, 2013). The latex of C. procera has shown standard solutions (0.05-0.35mg/ml) as described by
larvicidal efficacy against all three important vector Slinkard and Singleton (1977) but with some
species viz, Ae. aegypti, An. stephensi and Cx. modifications. Each extract solution (0.1 mL) was mixed
Quinqefaciatus (Shahi et al., 2010). The hypoglycemic with 2 mL of a 2% (w/v) sodium carbonate solution and
property of C. procera Linn was also assesse by an oral vortexed vigorously. After 3 minute, 0.1 mL of 50% Folin-
glucose tolerance test (OGTT) in STZ-diabetic rats (Singh Ciocalteu’s phenol reagent was added and incubate for 30
et al., 2014). It is reported to contain cardiac glycosides, β- min at room temperature and then absorbance was
sitosterol, madrine, saponins, alkaloids, tannins, measured at 760 nm. All samples were analyzed in
trisecharoides, calotropon and flavonols (Edman et al., triplicates. Total phenolic contents of extracts were
1983). Calotropis is one of such important traditional expressed as mg Gallic acid equivalent (GAE)/gm dry
medicinal plants reported to be used for different clinical weight. All samples were analyzed in triplicates. The
indication. The main objective of the present study is to aluminium chloride colorimetric assay was used for total
investigate comparative efficacy of phytochemical analysis flavonoids determination, as described by Zhishen et al.
and antioxidant activity of methanolic extracts of (1999). Results were expressed as mg of quercetin
Calotropis gigantea and Calotropis procera. equivalent / g extract.

MATERIALS AND METHODS In vitro antioxidant activity

Chemicals The free radical scavenging activity of the methanolic leaf,
1, 1- diphenyl -2- picryl hydrazyle (DPPH) and flower and root extracts of Calotropis species was
quercetin were procured from Sigma Chemical Co. (St. determined by using various in vitro assays such as DPPH•
Louis, US); Gallic acid, ferrozine, TPTZ, DMSO, Folin assay, reducing power assay, FRAP assay and ferrous ion
Ciocalteu reagent, were purchased from Merck Ind. Ltd. chelating activity.
All other chemicals and reagents were of analytical grade.
Free radical scavenging activity
Collection of Plant material The free radical scavenging activity of each
Fresh parts including leaves, flower and root of C. extract was determined by the method of Blois, 1958. The
gigantea and C. procera were collected from botanical decrease in absorbance was measured at 517 nm and the
garden of H. M. Patel Statue, Vallabh Vidhyanagar and the percentage inhibition activity was calculated from; [(A0–
selected plant species ware identified by Dr. Rachana A1)/A0] x 100, Where A0 is the absorbance of the control,
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and A1 is the absorbance of the extract/ standard. IC50 was Sulphoxide (DMSO) and final volume of 250ml was made
calculated from equation of line obtained by plotting a by tap water. A batch of 25 larvae was used for each test
graph of concentration versus % inhibition. Vitamin C was and the tests were performed in replicates. Mortality was
used as a positive control. recorded after 24 hours. Dead larvae were identified when
they failed to move after probing with a needle in the
Reducing power siphon or cervical region. The experiments were conducted
The reducing power is determined by the Fe3+ – under laboratory conditions at 25–30°C and 80–90%
2+
Fe transformation in the presence of extracts. Reducing relative humidity. The 50% lethal Dose (LD50) was
power assay was determined according to the method of calculated.
Yildirim et al., (2001). Different concentrations of
methanolic extracts of the study species were mixed with Statistical analysis
1.0 ml of 200 mM sodium phosphate buffer (pH 6.6) and Significant differences of the data among the
1.0 ml of 1% potassium ferriccyanide followed by parameters were calculated by performing ANOVA test
incubation at 50°C for 20 minutes. After adding 1.0 ml of with the help of SPSS and means were compared by least
10% trichloro acetic acid, the mixture was centrifuged at significant difference (LSD). The values P < 0.05 were
3000 rpm for 10 minutes. The supernatant was taken out regarded as significant and the values P<0.01 were
and mixed with 2.0 ml of distilled water and 0.5 ml of 1% considered as highly significant.
ferric chloride. After incubation for 10 minutes, the
absorbance was measured at 700nm. Higher absorbance of RESULTS & DISCUSSION
the reaction mixture indicates reductive potential of the The present investigation provides a
extracts. All the tests were performed in triplicates and comprehensive profile of the antioxidant activity of
ascorbic acid was used as reference standard BHT. extracts of different plant parts of an important medicinal
plant, Calotropis species, with respect to its phenols and
Ferrous ion chelating activity flavonoids content. Our data shows significant antioxidant
The chelating of ferrous ions by methanolic potential exists, more importantly in the C. procera
extracts of the study species was estimated by the method compared to C. gigantea.
of Singh and Rajini (2004). The different concentrations of
methanolic extracts (leaf, flower and root) were mixed Phytochemical analysis
with 100μl of 2.0 mM ferrous sulphate solution and 300μl Phytochemical compounds were screened in
of 5.0 mM ferrozine. The mixture was incubated at room methanolic extract of leaves, flower and root of both
temperature for 10 minutes. The absorbance of the solution species C. gigantea and C. procera through qualitative
was measured at 562 nm. EDTA was used as standard. method presented in Table 1. Flavonoids and phenolic
Percentage of inhibition was calculated by using this compounds have been reported to possess antioxidant
formula, Percentage of inhibition = Absorbance of control activity in all parts of both Calotropis species. Form the
– Absorbance of test x 100 Absorbance of control result of phytochemical analysis, it is indicated that C.
procera showed the positive result for the presence of
Ferric Reducing Antioxidant Power (FRAP) cardiac glycosides but not C. gigantea except leaves. Table
In the FRAP assay, blue colored FeII- 1 show the presence and absence of various
tripyridyltriazine compound is formed from the colorless pharmacological active chemical constitute including
oxidized FeIII form by the action of electron donating alkaloids, saponins, phenols, sterols, flavonoids and
antioxidant. The change in absorbance was measured at terpenoids. Previous phytochemical studies on the aerial
593 nm. (Benzie and Strain, 1999) Briefly, 30µl standard parts of the plant showed the presence of alkaloids, cardiac
(Ferrous sulfate) or 50 µl sample was added to 1.5 ml glycosides, flavonoids, sterols and/or triterpenes reported
freshly prepared FRAP reagent (300mM acetate buffer, pH in C. procera as well as in C. gigantea (Mossa et al., 1991;
3.6;10 mM TPTZ in 40 mM HCL and 20mM FeCl3·6H2O Joshi et al., 2010).
in the ratio of 10:1:1). After 10 min incubation at 37°C, the
absorbance was read against at 593 nm. Results are Total phenolics and flavonoids content
expressed as µM ferrous sulfate equivalent per gram of The total phenolic content was calculated from
sample. standard curve of gallic acid with R2 value 0.98. The results
showed remarkably higher total phenolic content in the
Larvicidal bioassay leaves than flower and root extract in both of the species of
The larvicidal bioassay was done following the Calotropis. Root of C. gigantea (19.63 ± 1.3) and C.
standard World Health Organization protocols (WHO, procera (21.32 ± 1.85) shows similar total phenolic
2005). Insectory reared Ae. aegypti larvae were used for content. Flower and leaves of C. procera represent
the study. Test concentrations of methanolic extract significantly higher phenolic content than C. gigantea. Fig.
ranging from 100-1000 mg/l were prepared in Dimethyl 1 represents total phenolic and flavonoids content of
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leaves, flower and root of selected species of Calotropis. maximum metal chelating ability. There was no significant
The root extract of C. procera (28.13 ± 1.23) had shown difference in the IC50 value for metal chelating activity of
highest flavonoids content compared to leaves and all parts between C. procera and C. gigantea. Both species
followed by flower of Calotropis species. Significant exhibits chelating agents are effective as secondary
higher flavonoids content has been observed in C. procera antioxidants, because they reduce the redox potential there
compared to C. gigantea in all selected part of this plant. by stabilizing the oxidized form of the metal ion (Duh et
Phenolic compounds are known as powerful chain al., 1999).
breaking antioxidant. Several studies have described the The total FRAP activity of leaves, flower and root
antioxidant properties of medicinal plants which are rich in extract of both Calotropis species exhibits in Fig 4. FRAP
phenolic compounds (Pietta, 2000). Flavonoids are capable activity of C. procera leave extract was 784.34 ± 22.81
of effectively scavenging the reactive O2 species because mM FeSO4/ g powder which indicates the strong
of their phenolic hydroxyl groups and so they are potent antioxidant activity of the plant extract. Extract of the
antioxidants (Cao et al., 1997). flower also shown the higher activity of FRAP compared
to root extract. The root extract of C. gigantea demonstrate
In vitro antioxidant activity lowest FRAP activity compared to all other extract of the
The DPPH radical scavenging activity is a plant. Fig 4 indicate that C. procera possess comparatively
sensitive method for the antioxidant screening of plant higher antioxidant activity in reducing ferric ions than C.
extract. Extent of DPPH radical scavenged was determined gigantea. The reasons accounting for higher antioxidant
by the decrease in intensity of violet colour in the form of activity of methanol extract of C. procera compared to C.
IC50 values. In the present study, leave extract of C. gigantea in present investigation, might be due to
procera (84.23 ± 3.69) showed highest free-radical enhanced production of biologically active compounds
scavenging activity in term of % inhibition of DPPH like; alkaloids, tannins, saponins, and flavonoids etc. in this
radical compared to C. gigantea leaves (68.52 ± 2.71), but particular species (Elakkiya & Prasanna, 2012).
root extract of both Calotropis species showed very little The reducing capacity of a compound may serve
free-radical scavenging activity (Fig. 2). Lower IC50 value as a significant indicator of its potential antioxidant
represents higher antioxidant activity. The IC50 values of activity. There was concentration dependent increase in the
CPF, CGF, CGR, CPR, CGL, and CPL were found to be absorbance of reaction mixture for all the extracts and
0.68, 0.56, 0.42, 0.31, 0.32, and 0.21μg/ml respectively in standard. The results of reducing power assay represented
DPPH assay represent in Fig 3. Vitamin C used as a in form of EC50 value indicate conentration at which it
positive control and found to have an IC50 value 0.044 produced 50% absorbtion at 700 nm. CPL and CGL has
μg/ml. The leaves extract (CPL) of C. procera has lowest shown the maximum absorbance and hence maximum
IC50 value among the extracts and hence maximum reducing power among the extracts. Lower EC50 value of
antioxidant activity. Antioxidant activity of ethanolic C. procera vs C. gigantea of root (0.84 vs 1.02), leaf (0.42
extract of Calotropis gigantea has been reported in vitro by vs 0.5) and flower (0.71 vs 0.87) extract showed that C.
reducing power, DPPH and nitric oxide method (Joshi et procera have more potent reducing capacity (Fig. 5). The
al., 2010). significant linear correlation was confirmed between total
Significant lower IC50 value of DPPH radical phenolic, flavonoid content and FRAP, DPPH and
scavenging activity in C. procera than C. gigantea reducing activity (Khasawneh et al., 2011).
indicates more potent in scavenging free radical and Phytochemically, the plants have been
antioxidant activity might be due to higher content of investigated for cardenolides, cardiac glycosides from the
phenolic and flavonoid compound present in C. procera. latex and leaves, triterpenoids, anthocyanins from flowers
Most of the tannins and flavonoids are phenolic and pentacyclic triterpenes, alkaloid, cardinolides
compounds and may be responsible for antioxidant phytosterols and triterpenoid saponins from the root. C.
properties of many plants (Larson, 1988). procera and C. gigantea exhibit diversity in their chemical
The metal chelating ability of the methanolic constitute and secondary metabloites even witnin differernt
extracts was measured by the formation of ferrous ion part of the plant may be responsible for differnce in their
ferrozine complex. Each of the extract interfere with the antioxidant potential (Khairnar et al., 2012).
formation of Ferrous ion (Fe+2) and ferrozine complex
which suggests its metal chelating activity. IC50 for metal Larvicidal activity
chelating activity of leaves (0.98 and 1.24 mg/ ml), flower The results of larvicidal activity against Ae.
(2.5 and 2.9 mg/ ml) and root (2.38 and 2.98 mg/ ml) of C. Aegypti larvae are shown in Fig. 6 in the form of LC50
procera and C. gigantea were reported respectively, which value. The root and flower extract showed to be less toxic
was higher than the positive control EDTA with IC50 0.111 than leaves extract of C. procera and C. gigantea against
μg/ml (Fig. 3). Lower IC50 value represents higher Ae. aegypti. The LC50 values were 387 and 456 mg/l for
antioxidant activity. The leaves extract (CPL) of C. leaves extract of C. procera and C. gigantea, respectively
procera has lower IC50 value among the extracts and hence
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(Fig. 6). Overall, Ae. aegypti larvae were more susceptible in the LC50 value for larvicidal activity of all parts between
towards the leaves extract than flower or root extract of C. procera and C. gigantea which indicate both species
Calotropis species. Earlier studies of ethanolic extract of exhibits same effect in against Ae. aegypti larvae. An
C. gigantea have shown larvicidal activities on Ae. aegypti advantage of using botanicals as mosquito larvicide is due
larvae with LD50 value of 351.43ppm. The active to its photodegradative property as compared to chemical
principles in the Calotropis which might be have insecticides which causes toxicity in non-target organisms
insecticidal property (Shreya et al., 2012). Calotropin and and also causes environmental contamination. From the
Calotoxin might be main component responsible for the present it is clear that the leaves of both species of
larvicidal activities exhibits by Calotropis species (Dubey Calotropis formulation are highly effective as larvicide.
et al., 2007). There was no significant difference observed

Fig 1. Total phenolic and flavonoid content present in two Fig 2. Percentage Inhibition of DPPH radicals and metal
species of Calotropis procera and Calotropis gigantean chelating activity by various extract of both Calotropis
species

Fig 3. Inhibitory concentration IC50 value obtained from Fig 4. Ferric Reducing Antioxidant Power of methanolic
DPPH assay and metal chelating of various parts of extract of all studies parts of C. procera and C. gigantea
Calotropis procera and Calotropis gigantea

Fig 5. Reducing ability of different extract of leaves, Fig 6. LC50 value for larvicidal activity of various parts
flower and root of both Calotropis species in terms of EC50 of both Calotropis species
value
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Table 1. Phytochemical screening of Calotropis gigantea and Calotropis procera

Phytochemical Cardiac Terpenoids Steroids
Tannins Saponin Flavonoids Alkaloids
screening glycosides
Calotropis
gigantea
Flower - - + + - - -
Leaves + - + + + + +
Root - + + + - - +
Calotropis
procera
Flower - - + + + + -
Leaves + - + + + + +
Root + + + + + + +

CONCLUSION
The plant extract enriched with phenolic and procera possess maximum total antioxidant activity and
flavonoids can be used in routine life to treat various larvicidal activity than C. gigantea. The present
diseases which are due to free radicals generation in our investigation presents the first report on comparative
body. On the basis of the results obtained in the present analysis of antioxidant potential of extracts from its leaf,
study, it was concluded. Methanolic leaf extract of C. root and latex of C. procera and C. gigantea plants.

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