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Ultrasound-Assisted Extraction in Different Solvents for Phytochemical Study

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DOI: 10.2202/1556-3758.1599

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 International Journal of Food
Engineering
Volume 6, Issue 3 2010 Article 6

Ultrasound-Assisted Extraction in Different

Solvents for Phytochemical Study of Canna
indica
Padma S. Vankar∗ Jyoti Srivastava†

∗
Indian Institute of Technology, psv@iitk.ac.in
†
Indian Institute of Technology, jyoti@iitk.ac.in

Copyright 2010
c The Berkeley Electronic Press. All rights reserved.
 Ultrasound-Assisted Extraction in Different
Solvents for Phytochemical Study of Canna
indica
Padma S. Vankar and Jyoti Srivastava

Abstract

Red flowers of Canna indica (Family Cannacece) were extracted in Sonicator with different
solvents for the estimation of polyphenols and flavonoids using fresh as well as dry flowers. An-
tioxidant capacities of L- Ascorbic acid, trolox, pyrogallol and Canna flower extracts were also
evaluated based on its ability to scavenge the DPPH radical, results obtained were compared with
those of TEAC aay. A good correlation was observed between the two methods. The results
showed that acetone extract of fresh flowers contained the highest amount of antioxidant activity
78.33 %, 89.7 mg GAE / g and 51.45 µMT / g although the yield of the extract in the methanol
was highest (34 g / 100 g). Total phenol and flavonoids content (0.96 mg GAE / 100 g) (19.89 mg
QE / 100 g) were also evaluated for these extract. This is the first report on the phytochemicals
and antioxidant activity from Canna indica flowers particularly for its radical scavenging activity
and can be a good source of food additives.

KEYWORDS: Canna indica, polyphenols, flavonoids, antioxidant activity, radical scavenging

 Vankar and Srivastava: Phytochemical Study of Canna

1. INTRODUCTION

In recent years research interest has been growing widely to study the detailed
phytochemicals in medicinal plants. The plant based phytochemicals are classified
into various classes such as polyphenols, flavonoids, carotenoids, alkaloids,
tannin etc. Flavonoids are a class of low molecular weight, naturally occurring
polyphenolic compounds widely distributed in the plant kingdom. They exhibit
different biological functions that allow interactions between plants and their
environment. The phenolics present in flavonoids are also potent antioxidants
when tested in vitro [1]. Flavonoids like many other polyphenols are excellent
free radical scavengers (chain-breaking antioxidants) because they are highly
reactive as hydrogen or electron donors [2-6]. Flavonoids have been known to
offer protection against major diseases such as coronary heart diseases and cancer
[7] and they also exhibit biological activities, including antiallergenic, antiviral,
anti-inflammatory, antioxidant, antithrombotic, antiviral and anticarcinogenic
activities. However, most interest has been devoted to their antioxidant activity,
which is due to their ability to reduce free radical formation and also to scavenge
free radicals [8-10].
Today's toxic environment and modern life styles generate stress which
has enormous impact on our health, this increased stress culminates in formation
of large amount of free-radicals. These free radicals must be balanced or
deactivated to maintain good health. Due to biochemical processes, it is normal
for free radicals to be present in the body at all times. A normal healthy immune
system is normally able to control the existence of free radicals and minimize
their potential damage. Free radicals and other reactive oxygen species are
considered to be important causative factors in the development of diseases such
as neurodegenerative diseases cancer and cardiovascular diseases.
Interest in the role of antioxidants in human health has prompted research
in the fields of horticulture and food science to assess fruit and vegetable for their
antioxidant contents, such as ascorbate, carotenoids, tocopherols, and phenolics.
Natural antioxidants such as α-tocopherol and L-ascorbic acid are widely used
because they are seen as being safer and causing fewer side reactions, but their
antioxidant activities are, however, lower than those of synthetic antioxidants
such as butylated hydroxyanisole and butylated hydroxytoluene, tertiary butylated
hydroquinone and gallic acid esters. But synthetic antioxidants have been
suspected to cause or prompt negative health effects too. Hence, strong
restrictions have been placed on their application and there is a trend to substitute
them with naturally occurring antioxidants. Moreover, these synthetic
antioxidants also show low solubility [11].
Hence, there is a need for safe and economic viable sources of
antioxidants which have high activity from natural sources to replace these

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 International Journal of Food Engineering, Vol. 6 [2010], Iss. 3, Art. 6

synthetic antioxidants. The antioxidant compounds present in edible plants have

recently been promoted as food additives because they display little or no toxic
side effects.
Therefore, there is a growing interest in substances exhibiting antioxidant
properties that are supplied to human and animal organisms as food components
or as specific preventive pharmaceuticals. The plant kingdom offers a wide range
of natural antioxidants. However, there is still not enough knowledge about the
practical usefulness of most of them. In the group of secondary plant metabolites,
antioxidant phenolics are commonly found in various fruits, vegetables and herbs
and they have been shown to provide a defence against oxidative stress from
oxidizing agents and free radicals
Canna indica is a wildflower, commonly called "Indian shot" and it is
widely cultivated in India belongs to the family Cannaceae. Rhizome of Canna
indica has been used in traditional folk medicines. In the process of screening of
some plants, interest in red Canna flower (local name-Keli) led to this study. The
bright red color of this flower makes it a valuable ornamental plant and a potential
source for extraction of natural colorants for dyeing different types of textiles
[12].
In this study we have investigated the antioxidant activity in commonly
found red flower in India. We have compared Antioxidant activity in the flower in
different extracts paying special attention to this flower not previously analysed.
The aim of this paper was to evaluate the antioxidant properties of the
different extracts from Canna indica (crude methanol, ethyl acetate, acetone,
ethanol and water) and to correlate their antioxidant potential to the composition
of polyphenols.

2. MATERIALS AND METHODS

2.1 Chemical and spectral measurements: All the solvents used for extraction
were AR grade and for quantitative analysis HPLC grade were obtained from S.D
Fine chemicals- Gallic acid, Sodium carbonate, Folin-Ciocalteu (2N), Aluminium
chloride, Potassium persulfate, Sodium nitrite, Pyrogallol, Phosphate buffer,
Potassium ferricyanide, Trichloroacetic acid, Ferric chloride, DPPH (Alfa aser
95%), L- Ascorbic acid (Aldrich), Quercetin-98% (Hi Media), ABTS (Fluka),
Trolox (Aldrich), Distilled water (Millipore).

2.2 Plant Material: The red flowers of Canna indica was collected from I.I.T.
Campus, Kanpur, India and was identified at the C. S.A. Agricultural University,
Kanpur.

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 Vankar and Srivastava: Phytochemical Study of Canna

2.3.1 Conventional extraction: Extraction with organic solvents methanol,

acetone, ethyl acetate, ethanol were done in a soxhlet apparatus (borosil) for 10-
12 hrs and aqueous extraction is done on water bath in a beaker. Canna indica
fresh and dry petals were extracted in different solvents and water (100g in 400
ml of solvent each) at 600C temperature, the extracted material was recovered by
vacuo filtration through Whatman- 42 paper (Sortorius) in a Buchner funnel. In
all cases, the solvent was evaporated in a rotavapor (Buchi R-210) at 650C to near
dryness, and then dried in high vacuo for 24 h to achieve complete dryness and
the extract yield was determined as the increased weight of the flasks.

2.3.2 Ultra sonication-Assisted extraction: Each 20 g of flower was mixed with

50 ml of extracting solvents (Methanol, Ethanol, Ethyl-acetate, Acetone and
water) in a 100 ml conical (Tarsons) 4.0 cm in diameter. The ultrasound chamber
(JULABO, USR3) having a frequency of 20 using different sonic power (100–500
W) for different time intervals (15–120 min). The power level was set at the
maximum (level 9) and the temperature during the 1–2 hour extraction period was
stabilized at 25-30 0C during the extraction. For indirect sonication, the sample
(conical) was immersed in an ultrasound chamber at the half height of the solution
in the flask, operating at 20 kHz frequency. The temperature was controlled and
maintained at below 30 0C by periodical replacing water in the bath with cold one
(15 0C). The flask was taken out and cooled to room temperature by cooling
water. The flower extracts were filtered through Whatman No. 42 paper and the
solution was collected. The residue was taken back and extracted again in the
same conditions. The extracts of the twice-extraction were mixed. The ultrasound
power actually delivered to the extracting liquid by the sonic bath was determined
by the calorimetric method. This suggests that the Ultra sonication-Assisted
extraction process is more selective than the conventional one.

2.4 Total Phenolic Content: The total phenolic content (TPC) in the extract of
different solvents with dry and fresh flowers was determined using Folin –
Ciocalteu reagent [13]. To the 0.1 g of the dry extract 4.5 ml of de-ionized water,
0.5 ml filtrate, 0.2ml of 2N Folin-Ciocalteu reagent, 0.5ml of 2.5% sodium
carbonate and finally 4.3 ml of de-ionized water were added and mixed
completely. After 2 hours, the absorbance of the solution at 725 nm was measured
in triplicate in a spectrophotometer (Heλios α, Thermo Electron Corp.).
Quantification was based on the standard curve of Gallic acid (0-0.5 mg/ml),
which was dissolved in de-ionized water and expressed as Gallic Acid Equivalent
per 100 g extract.

2.5 Total Flavonoids Content: Total Flavonoids Content (TFC) of different

solvent extracts of Canna indica flowers was determined by Zhishen et al.,[14]. A

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 International Journal of Food Engineering, Vol. 6 [2010], Iss. 3, Art. 6

0.5 ml aliquot of the flower extract, (10mg/10ml), was mixed with 0.4 ml distilled
water, 0.15 ml of 5 % sodium nitrite was added and the mixture was allowed to
react for 5 min. Following this 0.15 ml of 10% Aluminium Chloride was added
and the mixture was made to stand for 5 min further. Finally, the reaction mixture
was diluted with distilled water and the absorbance at 510 nm was recorded in
triplicate against a blank. Total flavonoid content was calculated using Quercetin
as standard and expressed as mg Quercetin equivalents.

2.6 Determination of Antioxidant activity: Different solvent extracts of Canna

indica flowers often exhibit a strong antioxidant activity, notably due to the
presence of hydroxyl groups and aromatic heterocyclic rings that provide these
molecules with hydrophilicity and particular stability.

2.6.1 DPPH Assay: Antioxidant activity (AOA) assay was done by the DPPH
method described by Sanchez-Moreno (1990) [15]. The different extracts were
measured in terms of hydrogen donating or radical scavenging ability using a
stable radical DPPH. 2.8 ml of DPPH solution (0.045 mg/ ml) were rapidly mixed
with 0.2 ml and 0.4 ml of methanolic solution of plant extract (0.0025 g/10 ml)
one at a time in cuvette placed in the spectrophotometer. The absorbance at 515
nm was measured after 5 min. The initial absorbance of the DPPH was 1.2 -1.3.
The decline in radical concentration indicated the radical scavenging activity of
the sample. Pyrogallol was used as reference corresponding to 100% radical
scavenging activity. Radical scavenging activity was evaluated as percentage was
calculated as shown in equation (1):

(A0 - Atest) / (A0 - Aref ) X 100 Eq (1)

where A0 is the initial absorbance (DPPH + Sample) and Aref and Atest are
absorbance after 5 min with pyrogallol solution and sample solution. All
measurements were done in triplicate.

2.6.2 Trolox equivalent antioxidant capacity (TEAC) assay: Antioxidant

capacity of different extracts were evaluated by the improved ABTS *+ (2,2′-
azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) radical method as described by
Re et al., (1999) [16] with slight modification. ABTS *+ radical cation was
generated by a reaction of 7 mM ABTS with 2.45 mM potassium persulphate.
The reaction mixture was allowed to stand in the dark for 16 hrs at room
temperature and used within two days. The ABTS*+ solution was diluted with
deionized water to give an absorbance of 0.700 at 734 nm. All samples were
diluted appropriately to give absorbance values 20-80% of the blank. Trolox

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 Vankar and Srivastava: Phytochemical Study of Canna

solution was used as reference standard. The ABTS*+ scavenging effect (%) is
calculated as shown in equation (2):

(OD 734 standard - OD 734 sample)/ OD 734 standard) X 100 . Eq (2)

2.4.3 Evaluation of Antioxidant Activity in FRAP system: The Ferric Reducing

Antioxidant Power of Canna indica was evaluated according to Oyaizu, (1986)
[17]. Different extracts were suspended in distilled water and mixed with 2.5 ml
of 0.2 M phosphate buffer (pH 6.6), and 2.5 ml of 1% Potassium ferricyanide.
The mixture was incubated at 323 K for 20 min, 2.5 ml of 10 % Trichloroacetic
acid was added to the mixture and centrifuged at 3000 rpm for 10 min. The upper
layer of the solution (2.5 ml) was mixed with distilled water (2.5 ml) and Ferric
chloride (0.5 ml, 0.1 %), and the absorbance was measured at 700 nm. Increase in
absorbance of the reaction mixture indicated reducing power. The standard curve
was prepared using 2, 4, 6, 8, 10 mg l-1 solutions of Gallic acid in methanol: water
(50:50, v/v). Total phenol values were expressed in terms of Gallic acid
equivalent (mg g –1 of dry mass).

2.5 Statistical analyses: All the experiments were carried in triplicates and the
experimental results represent treatment groups expressed as means ± SD.

3. RESULT AND DISCUSSION

Canna indica flowers have been extracted with different solvents and then various
phytochemical analyses have been carried out with these extracts. The presence of
high content of polyphenols and flavonoids has been shown to be the cause of
good antioxidant activity in these extracts.

3.1 Optimization of ultrasonic extraction: In this work, a new extracting

method involving ultrasonic treatment was proposed. Previous studies have
shown that the Conventional extraction (Soxhlet extraction) of pigment in
different solvents is temperature dependant. The results of the Ultrasound
Assisted Extraction experiments indicated that ultrasound enhanced the efficiency
of Canna flower extraction by shortening the extract time and lowering the
volume of the solvents needed, but at the same time. This method did not increase
the yield very much on comparing with conventional solvent extraction. The
reason of this phenomenon might be due to the fact that the hydroxyl radicals
produced by acoustic cavitation of ultrasound in extracts, with the presentation of
a small amount of water in the fresh red Canna flowers. To fully investigate the
effect of the Ultrasonic efficiency of the extraction, the temperature of the
extraction mixture was reduced below its boiling point. This also produced a

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 International Journal of Food Engineering, Vol. 6 [2010], Iss. 3, Art. 6

pronounced increase in the color strength of the extract than the Conventional
extraction method. It can be observed from Fig 1 that Ultrasound Assisted
Extraction is better option in comparison with Conventional extractability.

3.2 Determination of yield: This is a first study on evaluation of phytochemicals

in red Canna indica flower which showed that among the various solvent-
methanol, acetone, ethanol, ethyl acetate and aqueous extracts of Canna indica.
The extraction yields for fresh and dry petals of red Canna, obtained employing
the selected solvents are shown in Fig 1. Ultrasonic Methanolic extract of fresh
Canna (MFC) flower showed highest yield 34.09 g/100g whereas as conventional
method showed yield 30.19 g/100g. Similarly, ultrasonic Ethyl acetate extract of
dry Canna (EADC) flower showed the lowest yield 7.24 g/100g whereas
conventional method showed 3.99 g/100g. Among all the solvents used in this
study, acetone extract of fresh flowers yielded second highest amount of crude
extract (20.43g/100g) which showed good and highest level of polyphenolics
TPC, TFC and AOA. This study demonstrates that these disadvantages of using
Soxhlet extraction method could be overcome by using ultrasound assisted
solvent extraction.

3.3 Determination of phenolic compounds in red Canna flowers: The phenol

concentrations in these organic solvent extracts and water extracts were found to
be high. It was determined from regression equation of calibration curve (y =
10.738x + 0.061, R 2= 0.98). The values of TPC varied between from 0.06 to 0.96
mg Gallic acid equivalent per 100 g of different solvent extracts as shown in table
1. The levels of total phenols determined in this way are not absolute
measurements of the amounts of phenolic materials but are in fact based on their
chemical reducing capacity relative to an equivalent reducing capacity of Gallic
acid. The different responses of the extracts in this assay may arise from the
solvent and variety of phenolics found in Canna. The Acetone extracts of both dry
(ACDC) and fresh (ACFC) had higher total phenolic compound (0.96 mg GAE/g)
as compared to other solvents and aqueous extracts.

3.4 Determination of flavonoids in red Canna flower: Flavonoids content were

expressed in (mg/g), in Quercetin equivalents. The samples showed values which
varied from 1.76 to 19.89. The highest amounts of flavonoids were found in
acetone extract of fresh (ACFC) flower of Canna, which contained the highest
amount of phenolics as well. It was observed that the amount of flavonoids
showed direct correlation with the total amount of phenolics. The high flavonoid
content in acetone extract of red Canna (ACFC) contributes largely to its
increased antioxidant potential in comparison to other extracts. Table 2 shows the

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 Vankar and Srivastava: Phytochemical Study of Canna

values of total phenolic contents and total flavonoid contents in ten different
extracts.

3.5 Determination of antioxidant activities in red Canna flowers: The acetone

fresh flower extract possesses significantly high antioxidant activity and is most
potent extract which showed good free radical scavenging effect as assessed by
the DPPH, TEAC and FRAP assays whereas the ethanolic dry flower extract
showed the weakest antioxidant activity. Different solvent extracts showed
different antioxidant activity due to the difference in concentrations of
anthocyanin, total phenols and flavonoids which got extracted from the dry and
fresh flowers of Canna indica. Hence, two methods have been used to measure
their antioxidant activities. Furthermore, these methods are reliable and easy to
perform and can be used for determining hydrophilic and lipophilic antioxidant
capabilities of samples. Results suggest that the red fresh flowers of Canna indica
could be a potential candidate for natural antioxidant

3.5.1 Total antioxidant capacity of Canna flower by ABTS *+ decolorization

assay: This method measures the relative antioxidant ability of flower to
scavenge the radical ABTS *+ the aqueous phase, as compared with a standard
amount of Trolox. The ABTS *+, generated by potassium persulfate, is presented
as an excellent tool for determining the antioxidant activity of hydrogen-donating
antioxidants (scavengers of aqueous phase radicals) and of chain breaking
antioxidants (scavengers of lipid peroxyl radicals). The antioxidant defense
system of the body is composed of a mixture of antioxidants. ABTS
decolorization assay has been used for the first time to evaluate the antioxidant
capacity of different extracts of Canna indica flower. As shown in Table 2, total
antioxidant capacity, in TEAC of flower tested, was found to range from 6.67-
51.45 μMT/g. The activity of the acetone (ACFC) flower extract was found to
show highest free radical scavenging effect while the ethanolic extract of dry
flowers (EDC) showed the lowest antioxidant activity. The results demonstrate
that Canna indica extract possesses an interesting antioxidant activity and is a
potential candidate for natural antioxidant.

3.5.2 Antioxidant activity by DPPH free radicals: All the extracts were studied
for antioxidant properties by DPPH (2,2-diphenyl-1-picrylhydrazyl) assay as well.
The efficiency of each extract differed depending on the particular solvent type
and methodology involved in total antioxidant capacity. The study revealed that
the extracts showed good antioxidant activity by this assay. The AOA of extract
of acetone (ACFC) showed the highest inhibition towards DPPH radicals
(78.33%) with respect to the control value by 50% (IC50). These results
demonstrated similar trend as the ABTS assay in antioxidant activity.

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 International Journal of Food Engineering, Vol. 6 [2010], Iss. 3, Art. 6

3.5.3 Determination of Reducing power by FRAP Assay: There is a wide range

of antioxidant activities within the extracts analyzed, as measured by the FRAP
(ferric reducing ability of plasma) method. The values of absorbance at 700 nm
for the ten crude extracts in different solvents of Canna flowers revealed that all
samples had a capacity to reduce iron (III), and the Reducing Power (RP) values
of the samples were significantly different, ranging from the lowest value of 11.9
(EDC) to the highest value of 89.7 mg GAE/g (ACFC). Based on the antioxidant
data obtained, Extract with FRAP antioxidant capacity high values will be further
investigated in the future to determine which chemical groups are responsible for
such high antioxidant capacity values.

3.6 Correlation of ABTS assay and DPPH assay: L- Ascorbic acid (AA) is one
of the most effective antioxidants in fruits and vegetables. People with high
intakes of dietary AA acid or citrus fruits have repeatedly been associated with
lowered risk of developing cancer. The AA contribution to scavenge ABTS *+
varies extensively up to 200 times the lowest value. A linear correlation between
reduced ABTS *+ and DPPH after different concentrations of AA were added.
Similar results were obtained when Trolox and Pyrogallol were added, and their
linear correlation could be described as [ABTS *+] = 0.963[DPPH] (R2 = 0.9993),
[ABTS *+] = 0.9987 [DPPH] (R2 = 0.9989) and [ABTS *+] = 1.688 [DPPH] (R2 =
0.9989) respectively. In this study, it is found that 1 mol of AA reacts with
approximately 2 mols of ABTS *+ radicals or DPPH· radicals. 1 mol of AA
reduces 2 mols of ABTS *+ radicals similarly, 2 mols of ABTS *+ or
DPPH·radicals are scavenged by 1 mol of trolox. The stoichiometry of the
reaction between pyrogallol and ABTS *+ or DPPH· radical is found to be 1:5 and
1:4, respectively. For the antioxidants tested, the stoichiometry of reactions
between antioxidant and ABTS *+ or DPPH· is very close. However, when
pyrogallol was used, the stoichiometry differs. The total antioxidant potential of
Canna flowers showed significant similarity by employing TEAC and DPPH
scavenging assay. However, the results obtained in this study, show a good
correlation between the two models.

4. Conclusions

The Ultrasound Assisted Extraction method reported here can offer an effective
alternative for simultaneous extraction of pigment (phytochemicals) from Canna
indica. The optimization process involved studying the response of the
experimental conditions is best and it is proved to be an effective method for the
extraction. Results obtained indicated that all the extracts from Canna indica
have a great potential as a source of natural antioxidant agent, which contain
fairly high amount of flavonoid and phenolic compounds, which is exhibited

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 Vankar and Srivastava: Phytochemical Study of Canna

through good antioxidant activity. The high scavenging property of flower

extracts may be due presence of large contents of phenolic compounds, the
hydroxyl groups present in the molecules can provide the necessary component as
a radical scavenger.

Table 1 Total Phenolic Contents and Total Flavonoid Contents in different

extracts

Extract TPC(mg GAE/100g) TFC(mg QE/100g)

MFC 0.54 ± 0.15 8.95 ± 0.09

MDC 0.15 ± 0.03 2.18 ± 0.04
AFC 0.34 ± 0.08 4.58 ± 0.06
ADC 0.10 ± 0.03 2.01 ± 0.04
EAFC 0.78 ± 0.17 12.44 ± 1.45
EADC 0.29 ± 0.05 6.89 ± 0.06
ACFC 0.96 ± 0.19 19.89 ± 1.79
ACDC 0.37 ± 0.11 9.85 ± 1.00
EFC 0.31 ± 0.06 4.00 ± 0.05
EDC 0.06 ± 0.01 1.76 ± 0.02

*MFC-Methanolic fresh canna and MDC-Methanolic dry canna

AFC-Aqueous fresh canna and ADC-Aqueous dry canna
EAFC-Ethyl acetate fresh canna and EADC-Ethyl acetate dry canna
ACFC-Acetone fresh canna and ACDC-Acetone dry canna
EFC-Ethanolic fresh canna EDC-Ethanolic dry canna

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 International Journal of Food Engineering, Vol. 6 [2010], Iss. 3, Art. 6

Table 2 Radical Scavenging Capacity of different samples of Canna indica

by three different methods

Extract DPPH(%Inhibition) FRAP(mgGAE/g) TEACµMT/g

MFC 37.00 66.6 ± 2.1 14.96 ± 0.15
MDC 11.80 35.3 ± 0.7 11.54 ±1.45
AFC 18.70 58.5 ± 1.5 12.02 ±0.51
ADC 7.00 21.2 ± 0.5 8.56 ± 0.23
EAFC 54.09 70.7 ± 2.7 20.22 ± 0.15
EADC 27.66 43.4 ± 0.8 16.78 ±1.45
ACFC 78.33 89.7 ± 2.9 51.45 ± 0.98
ACDC 44.68 62.6 ± 1.7 43.56 ± 0.67
EFC 16.98 51.5 ± 0.8 10.18 ±1.78
EDC 5.78 11.9 ± 0.5 6.67±1.45

*C-Ex- Conventional extraction; U-Ex- Ultrasonic extraction

Figure 1: Percentage Yield in different solvent Ultrasonic assisted extracts

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