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International Journal of Advances Drug Delivery
Volume 2, Number 1-2, January- December 2012, pp. 1-9
@ Research Science Press, (India)
1
In-vitro Antioxidant Activities and Total Phenolic Content
of the Ethanolic Leaf Extract of Croton Sparsiflorus
Growing in Bangladesh
Hemayet Hossain*, Abdullah-Al-Mansur1, Altaf Hossain**, Md. Sariful Islam Howlader***,
Shubhra Kanti Dey****, Arpona Hira****, Arif Ahmed****
* Senior Scientific Officer, Chemical Research Division BCSIR Laboratories, Dhaka, Bangladesh Council of
Scientific and Industrial Research, Dr. Qudrat-E-Khuda Road, Dhaka-1205, Bangladesh
**Pilot Plant and Process Development Centre, Bangladesh Council of Scientific and Industrial Research, Dr.
Qudrat-E-Khuda Road, Dhaka-1205, Bangladesh
***Department of Pharmacy, World University of Bangladesh, Dhaka-1205, Bangladesh
****Pharmacy Discipline, Life Science School, Khulna University, Khulna-9208, Bangladesh
E-mail- hemayethossain02@yahoo.com
Abstract:-The crude ethanolic leaf extract of Croton sparsiflorus Linn. (Family: Punicaceae) was evaluated for its possible
antioxidant activities growing in eastern part of Bangladesh. Five complementary test systems, namely DPPH free radical
scavenging, nitric oxide scavenging, reducing power, Fe++ ion chelating ability and total phenolic content were used for
determining antioxidant activities of the leaf extract. In DPPH free radical scavenging test, IC50 value for ethanolic crude extract
was found fairly significant 58.45±0.32 µg/ml while compared to the IC50 value of the reference standards ascorbic acid and
Butylated Hydroxy Anisole (BHA) (5.23±0.34 and 6.15±0.27 µg/ml) respectively. The ethanol extract showed maximum nitric
oxide (NO) scavenging activity of 68.02% at 100 µg/ml, where as ascorbic acid exhibited 88.43% inhibition at the same
concentration. The IC50 value for nitric oxide scavenging test was also found significant (75.09±0.58 µg/ml) while compared to
the IC50 value of the reference standard ascorbic acid (31.38±0.47 µg/ml). The maximum absorbance for reducing power assay
was found to 0.788 when compared to 2.817 and 2.031 for standard ascorbic acid and BHA at 100µg/ml respectively. In Fe++ ion
chelating ability test, the IC50 value of the extract was determined as 89.72 µg/ml where that of EDTA showed 8.75 µg/ml. The
total phenolic amount was also calculated as quite high in ethanolic crude extract 187.65 mg of gallic acid equivalent per gram of
dry extract. Therefore, the obtained results tend to suggest the antioxidant activities of the crude ethanolic extract of the leaves of
C. sparsiflorus and justify its use in folkloric remedies.
Key words: Croton sparsiflorus, DPPH free-radical scavenging, Nitric oxide scavenging, Reducing power, Total phenolic.
Introduction
Croton sparsiflorus (Family- Euphorbiaceae) is a small annual herb, growing up to 1-2 ft tall. Croton
sparsiflorus (C. sparsiflorus) is a widely distributed in Indian subcontinent from Punjab in the north to Madras in the
south and Bangladesh. This plant contains main chemical constituents like glycosides saponins, tannins, flavonoids,
terpenoids and alkaloids1. Three proaporphine bases designated crotsparine, N-methyl-crotsparine and N,O-
dimethylcrotsparine; two dihydroproaporphines, crotsparinine and N-methylcrotsparinine and aporphine alkaloid
have been isolated from C. sparsiflorus 2. The plant also contains phenolic and flavonoid compounds 3. The
powdered leaves are useful in controlling high blood pressure, and for the treatment of skin diseases and cuts
and wounds 4-6. The presence of quercitol also reveals that the C. Sparsiflorus leaves are used as antiseptic and
antidote4-6. Traditionally the leaves are used in the treatment of diarrhoea and dysentery. The alkaloid fraction of the
extract of C. sparsiflorus exhibits anti-inflammatory and anti-pyretic activity7. The leaves of the C. sparsiflorus have
been also exhibited antibacterial activity 8.
Cancer and atherosclerosis, two major causes of death, are salient "free radical" diseases in human.
Reactive oxygen species (ROS) have a tendency to donate oxygen to other substances. Many such reactive species
are free radicals and have a surplus of one or more free-floating electrons rather than having matched pairs and are,
therefore, unstable and highly reactive includes the hydroxyl radical (OH.), the superoxide radical (O.2), the nitric
oxide radical (NO.) and the lipid peroxyl radical (LOO.) cause severely deleterious effects on the human body 9.
Enzymatic and non-enzymatic reactions like respiratory chain reaction, the phagocytosis, prostaglandin synthesis,
cytochrome P450 system and oxidative phosphorylation (i.e. aerobic respiration) in the mitochondria10. ROS are the
products of normal cellular metabolism, having both deleterious and beneficial effect in the body 11. The balance
International Journal of Advances Drug Delivery
Volume 2, Number 1-2, January- December 2012, pp. 1-9
@ Research Science Press, (India)
2
between the production of free radicals and the antioxidant defenses in the body has important health implications. If
there are too many free radicals produced and too few antioxidants, a condition of "oxidative stress" develops which
may cause chronic damage body11. Antioxidants play an excellent role in preventing cell damage. They donate their
own electrons to free radicals. Free radical accepts the electron from antioxidant and they do not attack the cell and
the chain reaction of oxidation is inhibited 12. Phenolic compounds, flavonoid and triterpenoids containing foods and
beverages with antioxidant activity have been reported13. Very recently, health risks and toxicity have been reported
using synthetic antioxidants restricted14. Some well known natural antioxidants like rosemary and sage are already
exploited commercially either as antioxidant additives or as nutritional supplements stipulating the antioxidant
potential of plant species15. In recent years, the interest in natural antioxidant, especially of plant origin, has greatly
increased16.
Since there is no sufficient data currently available to substantiate antioxidant activities from leaf extract of
Croton sparsiflorus that abundantly growing in southeast part of Bangladesh, therefore the present study was
designed to provide scientific evidence for its use as a traditional folk remedy by investigating the antioxidant
activities that also confirm its use in pathological conditions where free radicals are implicated.
Materials and methods
Collection and identification of plant material
The leaves of C. sparsiflorus (Family- Euphorbiaceae) were collected from Chiattagong, Bangladesh in
December, 2011 and identified by Bangladesh National Herbarium, Mirpur; Dhaka (Accession No: 34405).
Preparation of ethanolic extract
The leaves of C. sparsiflorus were freed from any of the foreign materials. Then the roots were air-dried
under shed temperature followed by drying in an electric oven at 40º C. The dried plant materials were then ground
into powder. About 250g of powdered material was taken in a clean, flat-bottomed glass container and soaked in
1000ml of 95% ethanol. The container with its contents was sealed and kept for a period of 10 days accompanying
occasional shaking and stirring. The whole mixture then underwent a coarse filtration by a piece of clean, white
cotton material. Then it was filtered through whatman filter paper (Bibby RE200, Sterilin Ltd., UK) which was
concentrated with rotary evaporator at bath temperature not exceeding 40˚ to have gummy concentrate of extract
(yield approx. 2.77%).
Phytochemical screening
The freshly prepared crude extract was qualitatively tested for the presence of chemical constituents, by
using the following reagents and chemicals, for example, alkaloids were identified by the Dragendorff’s reagent,
flavonoids with the use of Mg and HCl, tannins with ferric chloride and potassium dichromate solutions, and
steroids with Libermann-Burchard reagent and reducing sugars with Benedict’s reagent17-19.
Chemicals
1,1-Diphenyl-2-picryl hydrazyl (DPPH), L-ascorbic acid, Butylated Hydroxy Anisole (BHA), Gallic acid,
Folin-ciocalteu phenol reagent, Ferrozine and Griess reagent were obtained from Sigma Chemical Co.[(St. Louis,
MO, USA)]. Trichloroacetic acid (TCA), Phosphate buffer (pH 6.6), Potassium ferricyanide [K3Fe(CN)6], FeCl2,
FeCl3, Sodium nitroprusside, Ethanol, Sodium phosphate, EDTA, Ammonium molybdate and Sodium carbonate
were of analytical grade and purchased from Merck (Darmstadt, Germany).
Total phenolic content determination
The modified Folin-Ciocaltu method20 followed to determine the total phenolic content of the extract of
leaves of C. sparsiflorus. A 0.5 ml of each extract (1 mg/ml) was mixed with 5 ml Folin-Ciocaltu reagent (1:10 v/v
distilled water) and 4 ml (75g/l) of sodium carbonate and the mixture was then vortexed for 15 second for the
development of color the mixture was allowed to stand for 30 min at 40°C.Then the absorbance was read at 765 nm
with the same spectrophotometer. Total phenolic content was calculated as mg of Gallic acid equivalent per gram
using the equation obtained from a standard gallic acid calibration curve y=6.2548x -0.0925, R2=0.9962.
International Journal of Advances Drug Delivery
Volume 2, Number 1-2, January- December 2012, pp. 1-9
@ Research Science Press, (India)
3
Antioxidant activities
DPPH free radical scavenging activity
The method of Chang et al 21 was used for performing the DPPH radical scavenging activity. A stock
solution (5mg/ml) of ethanolic extract of leaves of C. sparsiflorus (5 mg/ml) was prepared in respective solvent
systems. A serial dilutions were the carried out to obtain concentrations of 5, 10, 20, 40, 60, 80, 100 µg/ml. An equal
amount of sample solution was mixed with an equal amount of 0.1 mM methanolic solution of DPPH, The mixture
was vortex and allowed to stand at the dark at 25 °C for 30 min. After 30 min incubation, the absorbance of the
mixture was read against a blank at 517 nm using a double beam UV/Visible spectrophotometer (Analykjena, Model
205, Jena, Germany). The radical scavenging activity was expressed as the inhibition percentage (I%) and calculated
as per the equation:
I (%) = (Ablank – Asample / Ablank) x 100
Where Ablank is the absorbance of the control (containing all reagents except the test compound), and Asample
is the absorbance of the experimental sample with all reagents. IC50 value (the concentration of sample required to
scavenge 50% DPPH free radical) was calculated from the plot of inhibition (%) against the concentration of the
extract. All determination was carried out in triplicate and average of the results was noted. Ascorbic acid and BHA
was used as standard for this study.
Nitric oxide (NO) scavenging activity
Nitric oxide scavenging activity was measured spectrophotometrically22. Sodium nitroprusside (5 mmol) in
phosphate buffered saline was mixed with different concentrations of the extract of leaves of C. sparsiflorus (5–100
µg/ml) dissolved in methanol and incubated at 25 °C for 30 min. In control there was not test sample but an
equivalent amount of methanol was used. After 30 min, 1.5 ml of incubation the solution was taken and diluted with
1.5 ml of Griess reagent (1% sulphanilamide, 2% phosphoric acid, and 0.1% naphthylethylenediamine
dihydrochloride). The absorbance of the chromophore formed during diazotization of the nitrite with sulphanilamide
and subsequent coupling with naphthylethylene diamine dihydrochloride was measured at 546 nm. The nitric oxide
(NO) radical scavenging activity was expressed as the inhibition percentage (I%) and calculated as per the equation:
I (%) = (Ablank – Asample / Ablank) x 100
Where Ablank is the absorbance of the control reaction (containing all reagents except the test compound),
and Asample is the absorbance of the experimental sample with all reagents. IC50 value is the concentration of sample
required to scavenge 50% nitric oxide free radical and was calculated from the plot of inhibition (%) against the
extract concentration. All the determinations were carried out in triplicate and average of the absorptions was noted.
Ascorbic acid was used as positive control standard for this study.
Reducing power assay
The method of Dehpour et al23 was followed to determine the reducing power of C. sparsiflorus leaves of
ethanolic extract. Different concentrations of the extract (5-100 µg/ml) in 1 ml of distilled water were mixed with
phosphate buffer (2.5 ml, 0.2 M, pH 6.6) and potassium ferricyanide [K3Fe(CN)6] (2.5 ml, 1%). The mixture was
then incubated at 50°C for 20 min and a 10% solution of trichloroacetic acid (2.5 ml) was added to it. It was then
centrifuged at 3000 rpm for 10 min. The upper layer of the mixture (2.5 ml) was mixed with distilled water (2.5 ml)
and 0.5 ml, 0.1% FeCl3 and the absorbance of the mixture was measured at 700 nm with the same
spectrophotometer. Increased absorbance of the reaction mixture indicated increased reducing power. All the
determinations were carried out thrice and average of the results was taken. Ascorbic acid and BHA were used as
the standard reference compounds in this study.
Ferrous Ion Chelating Ability
The ferrous ions chelating activity of ethanol extract of leaves of C. sparsiflorus and standards were
investigated according to the method of Dinis et al24. Briefly, different concentrations of the extract (5-100 µg/ml)
were added to 0.1ml solution of 2 mM ferrous chloride (FeCl2). Then, the reaction was initiated by the addition of
International Journal of Advances Drug Delivery
Volume 2, Number 1-2, January- December 2012, pp. 1-9
@ Research Science Press, (India)
4
0.2ml of 5 mM Ferrozine and mixture was shaken vigorously and left standing at room temperature for 10 min.
After the mixture had reached equilibrium, the absorbance of the solution was then measured at 562 nm in
spectrophotometer, wherein the Fe+2 chelating ability of extracts was monitored by measuring the ferrous ion-
Ferrozine complex. The percentage of inhibition of ferrozine-Fe2+ complex formation was given in the below
formula:
Ferrous ions chelating ability (%) = [(A0 - A) /A0] × 100
Where A0 is the absorbance of the control solution (containing all reagents except extract); A is the
absorbance in the presence of the sample of plant extracts. All the tests were carried out in triplicate and EDTA was
used as standard.
Statistical Analysis
For antioxidant activity determination, data were presented as mean ± Standard deviation (S.D).
Results
Chemical group test
Results of different chemical tests on the ethanolic extract of C. sparsiflorus leaves showed the presence of
alkaloid, reducing sugars, steroid, tannins and flavonoids (Table 1).
Table 1: Results of different group tests of ethanolic extract of C. sparsiflorus leaves.
EE: Ethanol extract of C. sparsiflorus; +: Positive result; - : Negative result
Total phenolic content
The amount of total phenolic content was calculated as quite high in the ethanolic crude extract of C.
sparsiflorus (187.65±0.66 mg/g of gallic acid equivalent) (Table 2).
Table 2: Total phenolic content of the ethanol extract of C. sparsiflorus leaves.
Extract Avg. absorbance at
765 nm
Total phenolic content
mg of gallic acid equivalent (GAE)
per g of dry extract
Ethanol extract of
C. sparsiflorus leaves
1.151±0.07 187.65±0.66
The values are expressed as mean ± standard deviation (n=3).
Antioxidant activities
Ethanolic extract of C. sparsiflorus was screened for evaluation of its possible antioxidant activities. Five
complementary test systems, namely DPPH free radical scavenging, nitric oxide scavenging activity, reducing
power, ferrous ion chelating ability and total phenolic contents determination were followed for this analysis.
DPPH free radical scavenging activity
DPPH free radical scavenging activity of the C. sparsiflorus was found to be increased with the increase of
concentration of the extract (Table 3). The extract exhibited 69.87±0.66% radical inhibitions at 100 µg/ml whereas
at the same concentration the standards ascorbic acid and BHA exhibited 95.96 ±0.31% and 93.09 ±0.19%
inhibitions respectively. IC50 value of the extract was found to be very fairly significant (58.45±0.32 µg/ml) when
Plant
Extract
Alkaloid Reducing
Sugars
Tannins Gums Flavonoids Saponin Steroid
EE + + + - + + +
International Journal of Advances Drug Delivery
Volume 2, Number 1-2, January- December 2012, pp. 1-9
@ Research Science Press, (India)
5
compared to the IC50 value of the reference compounds ascorbic acid and BHA (5.23±0.34 and 6.15±0.27 µg/ml)
respectively.
Table 3: DPPH radical scavenging activity of the ethanolic extract of C. sparsiflorus leaves and standards.
Concentration
(µg/ml)
% Inhibition of extract and Standards at different concentration
Ethanol Extract of
C. sparsiflorus leaves
Ascorbic acid
(standard)
Butylated Hydroxyanisole (BHA)
(standard)
5 20.12±0.58 48.44 ±0.27 41.76 ±0.38
10 25.33±0.88 71.39 ±0.11 65.23 ±0.16
20 33.01±0.75 82.76 ±0.19 82.18 ±0.18
40 41.55±0.74 89.48 ±0.14 87.11 ±0.39
60 52.83±0.55 94.38 ±0.17 92.33 ±0.13
80 61.11±0.97 95.68 ±0.43 92.61 ±0.51
100 69.87±0.66 95.96 ±0.31 93.09 ±0.19
IC50 (µg/ml) 58.45±0.32 5.23±0.34 6.15±0.27
The values are expressed as mean ± standard deviation (n=3).
Nitric oxide (NO) scavenging assay
The scavenging of NO by the ethanol extract of C. sparsiflorus was increased in dose dependent manner.
Fig. 1 illustrates a significant decrease in the NO radical due to the scavenging ability of the extract and
ascorbic acid. The ethanol extract showed maximum scavenging activity of 68.02±0.38% at 100 µg/ml, where
as ascorbic acid at the same concentration exhibited 88.43±0.81% inhibition. The IC50 value for ethanolic
extract was found fairly significant (75.09±0.58 µg/ml) while compared to the IC50 value of the reference standard
ascorbic acid (31.38±0.47 µg/ml).
0
20
40
60
80
100
5 1020406080100
% Inhibition
Concentration (µg/ml)
Nitric oxide (NO) scavenging
activity
of P. granatum
P. granatum
Ascorbic acid
Fig. 1: Nitric oxide radical scavenging activity of the ethanolic extract of C. sparsiflorus leaves and standard. The
values are expressed as mean ± standard deviation (n=3).
Reducing power assay
In determination of reducing power of ethanolic crude extract of C. sparsiflorus, ascorbic acid and BHA
were used as positive control (Table 4). The maximum absorbance for ethanolic extract was found to be (0.788
±0.005) at 100 µg/ml concentration while compared to standard ascorbic acid (2.817 ±0.013) and BHA (2.031
±0.019) respectively, at the same concentration. With the increase of concentration, the absorbance of the extract
was found to be increased and those for the standards were also increased with increasing concentration.
International Journal of Advances Drug Delivery
Volume 2, Number 1-2, January- December 2012, pp. 1-9
@ Research Science Press, (India)
6
Table 4: Reducing power assay of the ethanolic extract of C. sparsiflorus leaves and standards.
Concentration
(µg/ml)
Average absorbance at 700nm of extract and Standards
Ethanol Extract of
C. sparsiflorus leaves
Ascorbic acid
(standard)
Butylated Hydroxyanisole (BHA)
(standard)
5 0.201 ±0.008 0.380 ±0.016 0.430 ±0.011
10 0.257 ±0.005 0.830 ±0.013 0.776 ±0.013
20 0.359 ±0.007 1.483 ±0.017 1.452 ±0.012
40 0.483 ±0.009 1.935 ±0.012 1.749 ±0.017
60 0.579 ±0.006 2.645 ±0.015 1.842 ±0.013
80 0.692 ±0.007 2.778 ±0.014 1.976 ±0.015
100 0.788 ±0.005 2.817 ±0.013 2.031 ±0.019
The values are expressed as mean ± standard deviation (n=3).
Fe++ ion chelating ability
Fe++ ion chelating ability of ethanol extract of leaves of C. sparsiflorus is shown in Fig. 2. The extract
showed 59.12±0.38% Fe++ ion chelating ability at 100 µg/ml where as the standard EDTA showed 99.87±0.15% at
the same concentration. The IC50 value of the extract was also found significant (89.73±0.44 µg/ml) while compared
to the IC50 value of the reference standard EDTA (8.75±0.27 µg/ml).
0
20
40
60
80
100
120
5 1020406080100
% Inhibition
Concentration (µg/ml)
Fe
2+
ion chelating ability of P. granatum
P. granatum
Ascorbic acid
Fig. 2: Fe2+ ion chelating ability of ethanol leaf extract of C. sparsiflorus and EDTA (Standard). The values are
expressed as mean ± standard deviation (n=3).
Discussion
A method based on the scavenging of the stable radical 1, 1-diphenyl-2-picrylhydrazyl (DPPH) has been
used extensively to predict the antioxidant activities of extracts of plants25- 26. The high inhibition value of C.
sparsiflorus ethanol extract may due to the presence of tannins and significant amount of flavonoids in the extract as
phytochemicals. Tannins and Flavonoids, commonly found in plants have been reported to have significant
antioxidant activity27.
NO scavenging capacity of the extract may help to arrest the chain of reactions initiated by excess
generation of NO that are detrimental to the human health. Nitric oxide is also implicated for inflammation, cancer
and other pathological conditions28. NO works as a atypical neural modulator that is involved in neurotransmitter
release, neuronal excitability and learning and memory. Besides its role in physiologic processes, it also
participates in pathogenic pathways underlying a large group of disorders including muscle diseases,
inflammatory bowel disease, sepsis and septic shock, primary headaches and stroke. Additionally, increasing
International Journal of Advances Drug Delivery
Volume 2, Number 1-2, January- December 2012, pp. 1-9
@ Research Science Press, (India)
7
evidence shows that NO modulates neurotoxin induced cell damage and is involved in neuronal cell death in
Parkinson’s disease (PD) and other neurodegenerative disorders such as Alzheimer disease29. Therefore,
antioxidants with free radical scavenging activities may have great relevance in the prevention and treatment of
diseases associated with oxidants or free radicals30. Preliminary phytochemical studies of the ethanol extract of C.
sparsiflorus showed the presence of tannins and significant amount of flavonoids therefore suppression of
released NO may be attributed to direct NO scavenging. Furthermore it has also been reported that the ethanolic
extract of C. sparsiflorus leaves possess apigenin & luteolin in where luteolin suppress nitric oxide production
possibly through reduction of inducible nitric oxide synthase (iNOS) enzyme expression31 and apigenin suppressed
COX-2 or inducible nitric oxide synthase (iNOS) expression as well as NO and PGE2 levels in lipopolyssacharide-
stimulated macrophages 32. Furthermore, the antioxidant potential of luteolin is twice stronger than that of vitamin
E33-34 and more potent antioxidant than the synthetic antioxidant butylated hydroxytoluene (BHT) 35. The presence
of ellagic acid in extract also exerts its antioxidant effects by inhibiting NADPH oxidase-induced overproduction of
superoxide, suppressing the release of NO by down-regulating iNOS36.
A direct correlation between antioxidant capacity and reducing power of certain plant extracts has been
reported37. The reducing properties are generally associated with the presence of reductones, which have been
shown to exert antioxidant action by breaking the free radical chain by donating a hydrogen atom38. The maximum
absorbance for the ethanolic extract of C. sparsiflorus was found to be 1.531 at 100 µg/ml while that of standard
ascorbic acid and BHA was found as 2.8111 and 2.031, respectively (Table 5).
Bivalent transition metal ions (e.g. Fe++) play an important role as catalysts of oxidative processes,
leading to the formation of hydroxyl radicals and hydroperoxide decomposition reactions via Fenton chemistry39.
These processes can be delayed by iron chelation. Iron can generate free radicals from peroxides and may be
implicated in human cardiovascular disease40. Therefore, minimizing its concentration affords protection against
oxidative damage. Ferrozine can quantitatively form complexes with Fe2+. The absorbance of Fe2+-ferrozine
complex was decreased dose-dependently, that is, the activity was increased on increasing concentration from
5 to 100 µg/ml. Table 6 is exhibiting the comparative percentage Fe++ ion chelating ability of ethanol extract and
standard compound (Na2EDTA). Therefore, the presence of flavonoids3 in the extracts also have scavenging activity
of free radicals generated by chelating activity with metal ions (e.g. Fe++) .
Phytochemical components, especially phenolic compounds (such as flavonoids, phyenyl propanoids,
phenolic acids, tannins etc.) are very important components for the free radical scavenging and antioxidant activities
of plants. Polyphenols are generally of the chemical patterns; phenolic groups react as hydrogen donors and
neutralize the free radicals29, 40. In the present study the total amount of phenolic compounds was calculated as quite
high in the ethanol extract of C. sparsiflorus leaves. The result of present study revealed that the presence of high
concentration of phenolic components in the extract might cause the high inhibition value of the extract. Phenols are
important components of plants. It is reported that the hydroxyl group of the phenolic compounds to eliminate
radicals and they contribute directly to antioxidant effect of the system.
Conclusion
In conclusion it can be revealed that the crude ethanolic extract of C. sparsiflorus leaves possess significant
antioxidant activity. The potential of the extract of C. sparsiflorus as antioxidant agents may be due to the presence
of phytoconstituents like polyphenols, tannin, flavonoids might be responsible for its activity and justify its use as a
traditional folk remedy. However, extensive researches are necessary to search for active principles responsible for
these activities.
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