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International Journal of Pharmaceutical Research

2012, Volume 4, Issue 2, 44-48

ISSN 0975-2366
Research Article

In vitro Antioxidant and Preliminary Phytochemical Studies of Caralluma fimbriata Wall.

Priya D1*, Rajaram K2,Suresh Kumar P2
Department of Biotechnology
Vel Tech High Tech Dr Rangarajan Dr Sakunthala Engineering College, Chennai- 600062
Anna University of Technology, Tiruchirappalli, Tamilnadu, INDIA
*Corresponding author E-mail ID:
Received: 17/06/2011, Revised: 15/07/2011, 25/05/2011
Preliminary phytochemical analysis and in-vitro antioxidant activity of aqueous, ethyl acetate, ethanolic & methanolic
extracts of Caralluma fimbriata Wall. were investigated. The antioxidant activity was studied in some in-vitro antioxidant
models like DPPH radical scavenging activity, superoxide radical scavenging activity, ferric reducing power. Total
antioxidant capacity was also determined by phosphomolybdate method. Among the phytochemical screening of these
extracts, methanolic extract showed that the whole plant was rich in alkaloids, flavonoids, glycosides, phenolic compounds,
saponins and quinones.The methanolic extract of Caralluma fimbriata Wall. showed antioxidant activity by inhibiting
DPPH, scavenging superoxide and hydrogen peroxide. It also showed reducing power ability in ferric reducing model.
Significant antioxidant activity of methanolic extract of Caralluma fimbriata Wall. was found which might be due to the
presence of Phenolic compounds. Flavonoids, Phenols, Saponins, glycosides found in the preliminary phytochemical
screening. The total phenols and flavonoid contents of aqueous, ethyl acetate, ethanolic & methanolic extracts Caralluma
fimbriata Wall. were also measured.
Key words: Antioxidant, Caralluma, DPPH, Apocynaceae, Phytochemical.
Free radicals contribute to more than one hundred
disorders in humans including atherosclerosis, arthritis,
ischemia and reperfusion injury of many tissues, central
nervous system injury, gastritis, cancer and AIDS [1, 2].
Oxidation process is one of the most important routs for
producing free radicals in food, drugs and even living
systems. Catalase and hydroperoxidase enzymes convert
hydrogen peroxide and hydroperoxides to nonradical forms
and function as natural antioxidants in human body. Due to
depletion of immune system natural antioxidants in
different maladies, consuming antioxidants as free radical
scavengers may be necessary [1, 3-5]. Recently there has
been an upsurge of interest in the therapeutic potentials of
medicinal plants as antioxidants in reducing such free
radical induced tissue injury. Also many other plant species
have been investigated in the search for novel antioxidants
[6-9] but generally there is still a demand to find more
information concerning the antioxidant potential of plant
species. But interest has increased considerably in finding
natural occurring antioxidants for use in foods or medicinal
materials to replace synthetic antioxidants which are being
restricted due to their side effects such as carcinogenicity
[10]. It has been mentioned the antioxidant activity of
plants might be due to their phenolic compounds [2].
Flavonoids are a group of polyphenolic compounds with
known properties which include free radical scavenging,
inhibition of hydrolytic and oxidative enzymes and antiinflammatory action [11]. Some evidence suggests that the
biological actions of these compounds are related to their
antioxidant activity [12]. Caralluma fimbriata Wall. an
edible succulent cactus is a perennial herb growing in dry
parts of Tamil Nadu, India. It belongs to the family
Apocynaceae is also a well known as Famine Food,
Appetite Suppressant & thirst quencher among tribal
population. Genus Caralluma comprises about 200 genera

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& 2500 species [13]. It grows wild all over India & is also
planted as a roadside shrub & boundary marker in gardens.
Several members of the genus Caralluma have found
medicinal uses in the treatment of Rheumatism, Diabetes,
Leprosy, Antiseptics & Disinfectants [14]. The species of
Caralluma found in India are edible and form part of the
traditional medicine system of the country. Caralluma
fimbriata is listed in The Wealth of India (1992) as
medicinal plant used as an appetite suppressant and has also
been used to treat diabetic, pain, fever, and inflammation.
Native Indian diets over many centuries have included
these edible wild succulent cacti, with claims in folklore
about its Appetite Suppressant Activity. An investigation
was carried out to find out the effect of Caralluma
fimbriata extract on appetite, food intake and anthrometry
in adult Indian men and women [15]. The extract of
Caralluma fimbriata in the form of capsules, has been
released under the trade name GENASLIM for body weight
The apocynaceae family members are mainly
distributed in the Himalayan, southern and western parts of
India. The plants chiefly inhabit arid soil. The plants were
collected from Pudukkottai (District), Tamil Nadu, India
and authenticated (Specimens No. BSI/SRC/5/23/0910/Tech-1569) in Botanical Survey of India, Coimbatore.
MS media, sucrose and all the chemicals for this study were
purchased from HiMedia, Mumbai, India. Glasswares were
purchased from Borosil, India.
Qualitative analysis of phytochemicals
Preparation for extracts
The plant was collected, washed and dried. Then it
was ground in a grinding machine to fine powder and

Priya et al / International Journal of Pharmaceutical Research 2012 4(2) 44-48

passed through a 24-mesh sieve and the extract is weighted
and stored at room temperature.
Extraction of plant material
The powdered sample (20g) of Caralluma fibriata was
successively extracted with 200ml of solvent (ethanol, ethyl
acetate and methanol) using magnetic stirrer and stirred for
3hrs. Then it was filtered using whatmann filter paper.
Again the residue was dissolved with 200ml solvent and
stirred for 2hrs. The solvent containing the extract is dried
under reduced pressure. The aqueous extract was prepared
with 10g of powder in 100ml of distilled water & stirred for
3 hrs. The supernatant was boiled up to minimum volume.
Phytochemical Screening
The freshly prepared crude extract was qualitatively
tested for the presence of biochemical constituents.
Qualitative Analysis. [16, 17]
Test for alkaloids
5ml of the extract was added to 2ml of HCl. To this
acidic medium, 1ml of Wagners reagent was added. A
reddish precipitate brown produced immediately indicates
the presence of alkaloids.
Test for glycosides
To a small amount of extract, 1ml of Fehling`s
solution was added and heated, orange precipitate indicates
the presence of glycosides.
Test for flavonoids
To 1ml of the extract, a few drops of dilute sodium
hydroxide was added. An intense yellow colour was
produced in the plant extract, which become colourless on
addition of a few drops of dilute acid indicates the presence
of flavonoids.
Test for saponins
The Extract was diluted with 20 ml of distilled water
and it was agitated in a graduated cylinder for 15 min. The
formation of 1cm layer of foam showed the presence of
Test for phenolic compounds
Small amount of various extracts were taken
separately in water and tested for the presence of phenolic
compounds with dilute ferric chloride solution. Violet color
indicates the presence of phenolic components.
Test for quinines
To a small amount of extract, concentration of
sulphuric acid is added. Appearance of red color indicates
the presence of quinones.
Test for reducing sugar
To few drops of the test solution, 2ml of Fehlings
reagent & 3ml of water is added. Appearance of Red orange
indicates the presence of Reducing sugar.
Quantitative analysis
Measurement of total phenolic contents:
Determination of total phenolic content was made
using Folin-Ciocalteaus phenol reagent [18]. Sample (1
ml), 0.5 ml of Folin-Ciocalteaus phenol reagent (2 N), and

2 ml of Na2CO3 (5%) were mixed and the reaction mixture

was allowed to proceed for 5 min at room temperature,
before dilution with 5 ml of deionized water. Each sample
was mixed thoroughly and placed in dark for 1 h and the
absorbance was measured at 725 nm with a UV-VIS
spectrophotometer. Tannic acid equivalent (mg/g) was
determined from a standard concentration curve. All tests
were performed in independent triplicates (n=3) and data
were expressed as mean SD.
Measurement of total flavonoid contents:
Flavonoid content was determined according to the
aluminum chloride colorimetric method [19] with some
modifications. Quercetin was used as a standard to make
the calibration curve. The sample solution (0.5 ml) was
mixed with 1.5 ml of 95% ethanol, 0.1 ml of 10%
aluminum chloride hexahydrate, 0.1 ml of 1 M potassium
acetate, and 2.8 ml of distilled water. After incubation at
room temperature for 40 min, the absorbance of reaction
mixture was measured at 415 nm. The same amount of
distilled water substituted for the amount of 10% aluminum
chloride as the blank. Using a seven point standard curve
(0-500 g/ml), the flavonoid content of extracts was
determined in independent triplicate (n=3).
Evaluation of in vitro antioxidant activity
DPPH radical scavenging assay:
The hydrogen donating ability of extracts was examined in
the presence of DPPH stable radical [20]. Sample stock
(1mg/ml) was diluted to final concentration 1,2,4,8,16
g/ml in ethanol. One ml of 0.3 mM DPPH ethanol was
added to 2.5 ml of sample solution of different
concentration and allowed to react at room temperature.
After 30 minutes the absorbance values at 517 nm were
converted into percentage. Antioxidant Activity (AA%)
using the following formula.
%Inhibition (AA) =

[(Abscontrol -Abssample)*100]

Ethanol (1.0 ml) plus plant extract solution (2.5 ml) and
DPPH solution (1 ml , 0.3 mM) was used as the test
solution ,DPPH solution (1 ml, 0.3 mM) plus ethanol (2.5
ml) was used as the control. The positive controls were
those using the standard (Ascorbic acid) solutions. The IC50
values were calculated by linear regression of plots where
the abscissa represented the concentration of tested plant
and ordinate the average percent of antioxidant activity
from mean of three separate tests. Experiment was repeated
in triplicated.
Ferric Reducing power ability:
The reducing power of the extract was evaluated
according to the method of Yen and Chen [21]. A volume
of 1 ml extract with distilled water, 2.5 ml of phosphate
buffer (0.2m, pH 6.6) and 2.5ml 1% potassium ferricyanide
was added and incubated at 50C for 30 minutes. After that
2.5 ml of Trichloroacetic acid (10%) were added to the
mixture and centrifuged for 10 mins at 3000 rpm, 2.5 ml
from the upper part were diluted with 2.5 ml of water and is
shaken with 0.5 ml fresh 0.1% ferrous chloride. The
absorbance was measured at 700 nm. The reference
solution was prepared as above, but contained water instead
of the samples increased absorbance of the reaction mixture
incubated increased reducing power.

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Priya et al / International Journal of Pharmaceutical Research 2012 4(2) 44-48

Evaluation of antioxidant capacity of phosphomplybdate method:
The total antioxidant capacity of the extract
determined with the phosphomolybdenum using tocopherol as the standard. An aliquot of 0.1 ml of the plant
extract (100 /ml) solution was combined with 1ml of
reagent (0.6M sulphuric acid, 28 mM sodium phosphate
and 4 Mm ammonium molybdate). The tubes were capped
and incubated in boiling water bath at 95 C for 90 mins.
After the samples had cooled to room temperature, the
absorbance of the aqueous solution of each was measured at
695nm against blank in UV spectrophotometer. The blank
solution contained 1 ml of reagent solution and appropriate
volume of same solvent used for the sample and it was
incubated under same conditions as rest of the sample. The
total antioxidant capacity was expressed as equivalents of
-tocopherol [10] liberated/L.
Estimation of superoxide scavenging activity
The Superoxide scavenging activity was performed by
Sanja method [22]. To the reaction mixture containing 0.1
ml of NBT (1 mg/ml solution in DMSO) and 0.3 ml of the
extract and standard in DMSO, 1 ml of alkaline DMSO (1
ml DMSO containing, 5mM NaOH in 0.1 ml water) was
added to give a final volume of 1.4 ml and the absorbance
was measured at 560 nm. Extracts (50-1000 g/ml) were
added to a hydrogen peroxide solution (0.6ml, 40mM). 300
l of plain DMSO, 0.1 ml NBT solution and 1 ml alkaline
DMSO was mixed and absorbance was taken at 560 nm and
this was taken as control reading. The percentage of super
oxide radical scavenging of Caralluma fimbriata extracts
and standard compounds was calculated as follows:
% Super oxide scavenging activity
Test absorbance Control absorbance
Test absorbance
Statistical analysis
Data were expressed as means standard deviation. The
inhibitory concentration, 50% (IC50), was calculated by
plotting the percentage of inhibition versus the
Qualitative Analysis of Phytochemicals
Qualitative analysis of phytochemicals were carried
out by Harborne & Kokate [16,17] method. Maximum
extraction of phytochemicals were found to be present in
Methanolic extract when compared with Ethanolic, Ethyl
acetate (EA) and aqueous extract of Caralluma fimbriata
(Table 1). The phytochemical screening of methanolic
extract showed that the whole plant was rich in alkaloids,
flavonoids, glycosides, phenolic compounds, saponins and
quinones. These compounds may be responsible for
medicinal activity as well as physiological activity of
Caralluma fimbriata.
Plant products including phenols, flavonoids, tannins
proanthocyanidins in the plants extracts have been reported
to be radical scavengers and inhibitors of Lipid
Peroxidation [23, 24]. The antioxidant properties of
phenolic acids and flavonoids are due to their redox
properties, ability to chelate metals and quenching of
singlet oxygen [25]. When Phytochemicals compounds
react with a free radical, it is the delocalization of the
gained electron over the phenolic antioxidant and the

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aromatic nucleus, that prevents the continuation of the free

radical chain reaction. This is often called Radical
Scavenging. But polyphenolic compounds inhibit
oxidation through a variety of mechanisms [26].
Quantitative Analysis of Phytochemicals
The total content of phenols in whole plant was found
to be 0.97% in methanol extract, 0.52% in ethanol extract,
0.4% in Ethyl acetate (EA) and 0.402% in aqueous extract
while flavonoids was found to be 0.5% in methanol extract,
0.39% in ethanol extract, 0.2% in ethyl acetate and 0.19%
in aqueous extract. Thus Quantitative estimation of
phytochemical constituents revealed that Caralluma
fimbriata contained highest yield of phenols and flavonoids
in methanolic extract
than ethanolic, EA and aqueous
extract ( Table2).The antioxidant activity was derived from
some flavonoid-type compounds, which are one of the most
diverse and widespread groups of natural phenolics [27]. It
is well known that plant flavonoids and phenols in general,
are the highly effective free radical scavengers and
antioxidants. Polyphenol and flavonoids used for the
prevention and cure of various diseases which is mainly
associated with free radicals. Thus the presence of
flavonoid in the plant extract as evident from
phytochemical screening may be responsible for the
Antioxidant activity.
Invitro Antioxidant activity of Caralluma fimbiata
DPPH radical scavenging activity
DPPH is a stable free radical that accepts an electron
or hydrogen radical and becomes a stable diamagnetic
molecule. A deep purple color with an absorption
maximum at 517 nm is formed from DPPH solution, but it
generally fades when some antioxidants are present in the
solution.The free radical scavenging activity of ethanol,
ethyl acetate, methanol and aqueous extracts of Caralluma
fimbiata were measured by the DPPH method. Methanol
extract exhibited maximum antioxidant activity (IC50:
50g/ml) than ethanolic (IC50: 200g/ml), ethyl acetate
extract (IC50: 900g/ml)
and aqueous extract
(IC50:235g/ml). However, -Tocopherol and ascorbic acid
which was used as positive control showed better radical
scavenging effect (IC50: 34.78
and 30.4 g/ml,
respectively) as shown in Fig. 1. Thus methanolic extract
had better reduction capacity of DPPH radical than
ethanolic,ethyl acetate and aqueous extract. In addition,
antioxidant activity had a linear relationship with the total
phenolic some plants [28]. In the case of Gymnema
sylvestre, decrease in the concentration of DPPH radicals
due to the scavenging ability of gymnema methanolic
extract. Maximum scavenging activity (57.10%) was
observed at 100 g/ml concentration and the IC50 value of
gymnema extract [29] were found to be 85.28 g/ml.
Reducing power ability
The measurements of the reducing ability of Fe3+ to
Fe transformation was investigated. The reducing power
indicates compounds that are electron donors, which can act
as primary and secondary antioxidants. The reducing power
of methanolic extract was increased with increasing amount
of sample (50, 100, 250, 500, 1000l/ml), but values
remained lower than that for ascorbic acid (Fig. 2).
Moreover, methanol extract had higher reducing activity
than ethanol, ethyl acetate and aqueous extract. The
reducing capacity of a compound may serve as a significant

Priya et al / International Journal of Pharmaceutical Research 2012 4(2) 44-48

indicator of its potential antioxidant activity .Therefore, the
higher phenolic content in ethanol extract might account for
the better results found in their reducing power. While, the
reducing power of the gymnema extract increased with
increasing the concentration and is comparable with the
standard ascorbic acid [29].

Superoxide scavenging activity

Superoxide radicals are known to be very harmful to
the cellular component. Super oxide free radical was
formed by alkaline DMSO which reacts with NBT to
produce coloured diformazan. The methanolic extract (IC50:
210g/ml) of Caralluma fimbriata scavenges superoxide
radical and thus inhibits formazan formation when
compared to ethanolic(IC50: 500
g/ml), ethyl acetate(IC50:
g/ml) and aqueous extracts(IC50: 840g/ml). Fig.4
illustrates increase scavenging of superoxide radicals in
dose dependent manner due to the scavenging
ability of the
Caralluma fimbriata methanolic extract. IC50 value of
ascorbic acid is 90 g/ml.
g/ml. A potent scavenger of free
radicals may serve as a possible preventative intervention
for the diseases [30 - 31].

Figure 1: DPPH radical scavenging activity

Concentration (g/ml)
Figure 4: Superoxide Scavenging Activity


Reducing Sugar
- Absence of Phytochemicals,
+, ++ Moderate of Phytochemicals,
+++ Presence of Phytochemicals


Antioxidant capacity by phosphomolybdate method

Methanolic extract had higher antioxidant capacity
than ethanol, ethyl acetate and aqueous extract, and this fact
might be associated with the relationship between the
antioxidant capacities with the molybdate
olybdate complex as
shown (Fig.3). Thus the results showed
owed that the four assay
methods were all suitable and reliable for assessing total
antioxidant capacities of plant extracts, although there were
some samples showing differences in total antioxidant
capacities between assay methods in the present study. The
total antioxidant capacity in the gymnema extract was
determined by the formation off the phosphomolybdenum
complex [29].





Concentration (g/ml)
Figure 2: Ferric reducing power ability


Table 1:: Qualitative Analysis of Phytochemicals






Table 2: Quantitative Analysis of Phytochemicals

Concentration (g/ml)
Figure 3: Antioxidant capacity by phosphomolybdate


Total Phenols %

Total Flavonoids %

Ethyl acetate



In this work, the maximum extraction of phytochemicals
were obsereved in methanolic extract than ethanolic, ethyl
acetate and aqueous extract. The total flavonoids &
phenolic compounds were found to be maximum in
methanolic extract than ethanolic, ethyl
acetate and aqueous
extract which reveals that Caralluma fimbriata is highly

IJPR | April June | 47

Priya et al / International Journal of Pharmaceutical Research 2012 4(2) 44-48

valuable in medicinal usage for the treatment of various
human aliments.
Authors are thankful to Dr.Rangarajan & Dr.Sakunthala
Rangarajan, Chairpersons of Vel Tech group of institutions,
Chennai and Anna University of Technology,
Tiruchirappalli, India for providing Technical support in the
form of instruments, encouragement and guidance during
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