In Vitro Phytochemical screening, Antioxidant, Antifungal and Cytotoxic activity of
methanolic extract of Centaurea pullata
Wasi Ullah Khan, *Rahmat Ali Khan, Mushtaq Ahmed, Huda Mohammad, Alkreathy, Latif
Ullah Khan, Mir Sadiq Shah
Department of Biotechnology, University of Science and Technology Bannu-28100, KPK,
Pakistan
*Corresponding author: Rahmat Ali Khan, Assistant Professor, Department of Biotechnology,
Faculty of Biological Sciences, University of Science and Technology Bannu KPK, Pakistan.
E mail: [email protected]
Telephone No: +92 51 90643086
Fax No: +92 51 9205753
Wasi Ullah Khan: Department of Biotechnology, University of Science and Technology Bannu28100, KPK, Pakistan
Email:[email protected]
Contact No, 03341913098
*Rahmat Ali Khan: Department of Biotechnology, University of Science and Technology
Bannu-28100, KPK, Pakistan
Email: [email protected] Contact No, 03145796104
Mushtaq Ahmed: Department of Biotechnology, University of Science and Technology Bannu28100, KPK, Pakistan
Email: [email protected]
Mir Sadiq Shah
Contact No, 03329977165
Huda Mohammad Alkreathy: Department of Pharmacy, King Abdul Aziz University Saudi
Arabia
Email:[email protected]
Latif Ullah Khan: Department of Biotechnology, University of Science and Technology Bannu28100, KPK, Pakistan
Email:[email protected] Contact No, 03319157749
Mir Sadiq Shah
Latif Ullah Khan: Department of Biotechnology, University of Science and Technology Bannu28100, KPK, Pakistan
Email:[email protected] Contact No, 03319157749
Abstract
Objectives: The main aim of this study is to find and extract the different types of biochemical
constituents and many other new antioxidants which should replace the synthetic one because of
some side effects and carcinogenic properties. Development of new potent antimicrobial agents
against multi drug resistant strain and cytotoxic assessment of the selected plant.
Methods: The Centaurea pullata medicinal plant was purchased during September 2014 from
local herbal store in District Bannu. Dried plant was ground and extracted with methanol to
prepare methanol crud extract. According to standard protocol the In-vitro biological activities
were conducted using the methanolic extract of plant. Cytotoxic activity of plant methanolic
extract against brine shrimps was done while antifungal activity was also measured.
Results: 100% death rate of brine shrimp was observed at 3mg/ml of plant extract after 72 hrs.
94±0.00% growth inhibition of Aspergillus fumagatus were noted during the present study.
Phytochemical screening were also done and results showed that plant possess different types of
phytochemicals. Significant scavenging results were observed during scavenging of free radicles
viz; 85.1% against DPPH, 83% to ABTS, 77% to Hydrogen peroxide and 93% against
phosphomolybedate, 83.23% t0 β - Carotene and 86% against Hydroxyl radical at 3mg/ml were
obtained.
Conclusion: Our findings provide evidence that Centaurea pullata roots showed its
effectiveness for free radicals scavenging that they may be used as a good antioxidants.
Considering the results of biological assays it can be concluded that most of the tested fractions
appeared as an important source for the discovery of new antimicrobial drugs and antitumor
agents not only for the therapeutic use of animals but also for humans.
Keywords:
Centaurea pullata, bioactive compounds, antioxidant activity, cytotoxic, antifungal activity,
DPPH, ABTS, β – Carotene.
Introduction:
Reactive oxygen species (ROS), such as superoxide anions, hydrogen peroxide, and hydroxyl,
nitric oxide and peroxynitrite radicals, play an important role in oxidative stress related to the
pathogenesis of various important diseases1. In healthy individuals, the production of free
radicals is balanced by the antioxidative defense system; however, oxidative stress is generated
when equilibrium favors free radical generation as a result of a depletion of antioxidant levels.
The oxidation of lipid, DNA, protein, carbohydrate, and other biological molecules by toxic ROS
may cause DNA mutation or/and serve to damage target cells or tissues, and this often results in
cell senescence and death. Cancer chemoprevention by using antioxidant approaches has been
suggested to offer a good potential in providing important fundamental benefits to public health,
and is now considered by many clinicians and researchers as a key strategy for inhibiting,
delaying, or even reversal of the process of carcinogenesis2. Already some natural antioxidant
(for example, rosemary and sage) are sold on business trade either as antioxidant flavors or
nourishing food supplements3. Similarly, many other plant classes have been reported in the
examine for novel antioxidants4, 5, 6, 7 but normally there is still a need to find more information
relating to the antioxidant activity of plant species8, 9 recommended that some nutrients and nonnutrient molecules of the medicinal and automatic plants show us antimicrobial properties. It can
also save us from different particular pathogens .According to WHO reports, more than 40%
deaths have been occurred due to infectious microbes in developing countries10. Medicinal plants
are so much valuable that it is also used in the treatment of cancer and it also plays a significant
part as a source of active anticancer agents11. Nearly more than 60% anticancer agents are used
in different ways from the natural source12. The discovery of the new antitumor drug with low
side effects on immune system has become a vital goal in many studies of
immunopharmacology13.The genus Centaurea had been widely used in folk medicine for
hundreds of years 14. Medicinal properties of Centaurea species are may be due to the presence
of acidic crystalline unsaturated lactones15. The Centaurea species had been used in traditional
medicine as diuretic, to treat diabetes and fever16.Centaurea species are well-known in folk
medicine to have anti-inflammatory, antirheumatic, anti-diarrheic, choleretic, cholagogue,
stomachic, menstrual, anti-dandruff, digestive, astringent, hypotensive, anti-pyretic, cytotoxic as
well as anti-bacterial effects and are used alone or mixed with other plants 17.
Material and Methods:
The Centaurea pullata medicinal plant was purchased during September 2014 from local herbal
store in District Bannu. and was recognized by Prof. Abdul Rahman, GPGC Bannu. Dried plant
ground mechanically up to mash size 0.1mm.100 g fine powder of Centaurea pullata was socked
in 500 ml methanol with temperate shaking and then placed it at room temperature for 5 days,
thus after the five days the plant is extracted and filtered by using what man filter Paper and
concentrated with the help of the rotary evaporator, after the concentration the extra methanol
was evaporated at 37оC to obtain crude extract.
Phytochemical screening was performed to recognize the existence of bioactive compounds in
plant part by using standard phytochemical methods as described by18.
Cytotoxic brine shrimp lethality test was carried out according to19 with some modification as
described20.
The antifungal activity of the plant extract was screened through the agar tube dilution method
by using the protocol by21.
Method was used for determination of DPPH scavenging ability of various fractions. 3 mg
DPPH was dissolved in 30 ml methanol to prepare stock solution22.The stock solution was
further diluted with methanol until reaching an absorbance less than 1.00 using the
spectrophotometer at 517 nm. Scavenging calculated through the following formula.
Scavenging effect (%) = [(OD of control-OD of sample) / (OD of control)] ×100
With certain modifications the ABTS radical scavenging assay was measured using the method
developed by 23.Equal volumes of 7 mM ABTS solution and 2.45 mM potassium per sulfate
solution were mixed to prepare stock solution and incubated in the dark for 12h at room
temperature to yield a dark colored solution consisting of ABTS•+ radicals. 50%methanol and
stock solution were mixed to prepare working solution for an initial absorbance of about0.700 (±
0.02) at 745nm, with control temperature set at 30°C. Free radical scavenging activity was
determined by mixing 100μl of different concentrations (3mg/ml, 1.5mg/ml,0.75mg/ml and
0.37mg/ml in methanol) with 1 ml of ABTS working standard. When the solutions were mixed
then after 1min the absorbance was measured. Experiment was done on four concentrations.
Ascorbic acid was used as positive controls in this experiment. The scavenging activity was
determined based on the percentage of ABTS radicals scavenged by the formula given below.
Percent scavenging = [(A0 −As) / A0] × 100
Where
A0 = absorption of control,
AS = absorption of sample solution.
With certain modifications the Hydrogen peroxide scavenging activity was assessed using the
method developed by 24.
The antioxidant activity of samples was calculated by the phosphor molybdenum method
according to the procedure of 25. An aliquot of 0.1 ml of sample solution was mixed with 1 ml of
reagent solution (0.6 M sulphuric acid, 28 mM sodium phosphate and 4 mM ammonium
molybdate). The test tubes were capped with silver foil and incubated in a water bath at 95 ºC for
90 min. After the samples had cooled to room temperature, the absorbance of the mixture was
measured at 765 nm against a blank. Ascorbic acid was used as standard. The antioxidant
capacity was estimated using following formula:
Antioxidant effect (%) = [(control absorbance-sample absorbance) / (control absorbance)] ×100.
Hydroxyl radical scavenging activity of extracts was examined by the method of 1. The reaction
mixture contained 50 ml of 2-deoxyribose (2.8 mM) in phosphate buffer (50 mM, pH 7.4), 10 ml
of premixed ferric chloride (100 mM) and EDTA (100 mM) solution (1:1; v/v), 10 ml of H2O2
(200 mM) without or with the extract solution (10 ml). The reaction was triggered by adding 10
ml of 300mMascorbate and incubated for 1h at 37 °C. A solution of TBA in 1 ml (1%; w/v) of
50mMNaOH and 1 ml of 2.8% (w/v; aqueous solution) TCA was added. The mixture was heated
for 15 min on a boiling water bath and then cooled. The absorbance was measured at 532 nm.
The scavenging activity on hydroxyl radical was calculated as follows:
Scavenging activity (%) = (1- absorbance of sample / absorbance of control) × 100
The assay was performed as given by 26 and modified slightly. First, 1mg of β-carotene dissolved
in 5 ml of chloroform was mixed with 10 mg of linoleic acid and 50 ml of Tween80 followed by
chloroform removing under nitrogen and 25 ml of distilled water adding with vigorous shacking
to prepare β-carotene linoleate emulsion. An aliquot of sample (200 µl) was mixed with 800 µl
of the suspension, and then the absorbance was resolute at 470 nm at 45 °C for 2 h. β-Carotene
bleaching inhibition was estimated as the following equation:
Bleaching inhibition (%) = (β-carotene content after 2 h of assay/initial β-carotene content) ×
100
Results:
Centaurea pullata roots demonstrated that Phlobatannins, Tannins, Anthraquinone and
Terpenoids are present in the extract while Flavonoids and Saponins are found absent (Table 1).
The Centaurea pullata methanolic extract (CPME) shows its Antifungal activities up to some
extant against Aspergillus niger, Aspergillus flavius, Aspergillus fumigatus strain. The Centaurea
pullata methanolic extract (CPME) showed activity against Aspergillus flaviusi.e. 92% followed
by Aspergillus Niger (93%) while the highest activity was shown against Aspergillus fumegatrus
(94%). Likewise the terbinofine, a positive control was shown highly active against this fungal
strains, while the DMSO (negative control) shows zero percent (0%) inhibition activity against
all the used three fungal strains as shown in (Table 2).
After 24, 48 and 72hrs incubation the cytotoxic effects of extract of different concentrations were
noted and found that the brine shrimp survival rate is inversely proportional to the concentrations
of the plant extracts and the period of incubations. The results of the present study show that
Centaurea pullata possess significant cytotoxic activity as shown in (Figure 1).
For the matching of the antioxidant activity we used ABTS radical scavenging test which are
appropriate for both lipophilic and hydrophilic antioxidants. The ABTS (2,2, azo-bis-(3-ethyl
benzothiazoline-6-sulphonic acid) free radical scavenging capacity of the sample extract along
with the standard ascorbic acid was noted. It is found that the scavenging ability of the sample
extract is some less than the standard ascorbic acid as shown in (Figure 2).
To compare the antioxidant activity we used DPPH radical scavenging assay. The 1,1diphenyl2- picryl – hydrazyl (DPPH) free radical scavenging capability of the sample extract
Centaurea pullata along with the standard ascorbic acid noted on different concentrations. It was
found that the scavenging capability of the sample extract is some less than the standard ascorbic
acid as shown in (Figure 3).
The hydroxyl radical (•OH) negotiate membranes of cell at particular locations; react with most
biologically active macromolecules resulting in tissue damage and death of the cell. Therefore,
scavenging of •OH is essential to defend the living systems. The IC50 of •OH scavenging effect
by various fractions along with ascorbic acid. Thus the methanolic extract of Centaurea pullata
showed distinct scavenging activity, markedly scavenged the free radial and were the most
potent than reference chemical compound shown in (Figure 4).
To match the antioxidant activity we used Phosphomolybedate radical scavenging assay. The
Phosphomolybedate free radical scavenging ability of the sample extract Centaurea pullata
along with the standard ascorbic acid recorded on different concentrations. It is found that the
scavenging capacity of the sample extract is some less than the standard ascorbic acid as shown
in (Figure 5).
With concern to the β- carotene bleaching assay, the antioxidant activity of sample can be
classified as 3mg/ml > 1.5mg/ml > 0.75mg/ml > 0.37mg/ml. At 3 mg/ml, β- carotene bleaching
inhibitions
were
87.34,1.5mg/ml81.56,
0.75mg/ml76.34and
0.37mg/ml71.27.β-carotene
bleaching assay showed the dose response graph for all the concentrations ranging from 3mg/ml-
0.37mg/ml. It is found that the scavenging capacity of the sample extract is some less than the
standard ascorbic acid as shown in (Figure 6).
The hydroxyl radical scavenging activity can be categorized as 3mg/ml> 1.5mg/ml > 0.75mg/ml
> 0.37mg/ml. All results showed antioxidant activity in dose dependent manner at concentration
3mg/ml-0.37mg/ml. However the scavenging activity of all the concentrations were it is found
that the scavenging capability of the sample extract is some less than the standard ascorbic acid
as shown in (Figure 7).
Discussion:
Maximum number of plant constituents were shown in Methanol fractions of all the tested plant
sample .The above bioactive substances shows resistance against oxidative injuries of many
diseases
27
. In literature, many medicinal plant showed their power of antioxidant due to the
presence of flavonoids and alkaloids
27, 28
. The folkloric use of plant roots of Centaurea pullata
for treating infections may be justified by the charisma of anthraquinones as they possess
antimicrobial 29 and antiparasitic properties 30.
Oxidation is a necessary step for the energy production in the living things. However through
this oxidation process “the oxygen consumption during the normal metabolism” the free radicals
are produced through various enzymatic systems. In little amount the ROS is beneficial in
growth regulation and signal transduction but in increased amount the ROS produced oxidative
stress, and attack on many macromolecules i.e. DNA, protein and lipids 1. Our results shows
some similarities with the investigation of
31
reported that the medicinal plants have highly
scavenge the free radicals. The antioxidants potential of methanolic extract of this plant could be
due to the presence of phenolic and polyphenolic compounds in this medicinal plant which
reduce the free radicals which cause the oxidative stress. The results obtained by 32 also support
the results obtained from our experiments.
As we know that the synthetic drugs have side effects and there is some strains of microorganism
which are antibiotic resistant so due to this reason investigation of potent antimicrobial drugs
obtained from natural resources has been an objective of researchers and scientists. In our
present study the antimicrobial activities of Centaurea pullata results shows that the bacterial
and fungal strain are inhibited by these samples. Due to the presence of phenolic compounds in
medicinal plants, showed antimicrobial activities33 and the antimicrobial activities of the
medicinal plants are also due to the presence of bioactive compounds saponins 34.
The cytotoxic activity of the plant extract provides information about the anticancer and
antitumor potential of Centaurea pullata. Cytotoxic effect of the methanolic extract of
Centaurea pullata was determined by using brine shrimps lethality test. The result showed that
the brine shrimps survival is inversely proportional to the methanolic extract of the of Centaurea
pullata plant. It was reported that methanolic fraction of Arceuthobium oxycedri showed 100 %
cytotoxicity at high dose for brine shrimps which are related to the present result. The result of
our present study supports the traditional usage of the studied plant and suggests that methanolic
extract possess some bioactive constituents with antimicrobial and as well as anticancer disease
caused by the pathogens 18.
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Table 1: Phytochemical composition of Centaurea pullata:
Sample
CPME
Phlobatannins Anthraquinone Tannins Flavonoids Terpenoids Saponins
+
+
+
_
+
_
Table 2: Antifungal activity of Centaurea pullata methanolic extract (CPME) % Inhibition:
Fungal Strain
Aspergillus flavius
Aspergillus niger
Aspergillus
fumegatus
Mean±SD
Concentrations
3mg/ml
1.5mg/ml
0.75mg/ml
0.37mg/ml
3mg/ml
1.5mg/ml
0.75mg/ml
0.37mg/ml
3mg/ml
1.5mg/ml
0.75mg/ml
0.37mg/ml
Centaurea pullata
92
85
80
74
93
87
79
77
94
77
62
57
Terbinofine
100%
97%
90%
84%
100%
97%
89%
85%
100%
96%
88%
83%
Figure1: Cytotoxic effects of Centaurea pullata methanolic extract (% Death):
%Scavenging
CPME potential against ABTS
3mg/ml
1.5mg/ml
0.75mg/ml
0.37mg/ml
Concentrations
Ascorbic Acid
Extract
Figure 2. ABTS free radical scavenging of Centaurea pullata methanolic extract and
ascorbic acid.
%Scavenging
CPME Scavenging effect against DPPH
3mg/m;
1.5mg/ml
0.75mg/ml
0.37mg/ml
Concentrations
Ascorbic Acid
Extract
Figure 3. DPPH free radical scavenging of Centaurea pullata methanolic extract and
ascorbic acid.
%Scavenging
CPME potential against H2O2
3mg/ml
1.5mg/ml
0.75mg/ml
0.37mg/ml
Concentrations
Ascorbic Acid
Extract
Figure 4. H2O2 free radical scavenging of Centaurea pullata methanolic extract and
ascorbic acid.
%Scavenging
CPME potential against
Phosphomolybdate
3mg/ml
1.5mg/ml
0.75mg/ml
0.37mg/ml
Concentrations
Ascorbic Acid
Extract
Figure 5: Phosphomolybedate free radical scavenging of Centaurea pullata methanolic
extract and ascorbic acid.
%Scavenging
CPME Scavenging effect against β Carotene
3mg/ml
1.5mg/ml
0.75mg/ml
0.37mg/ml
Concentrations
Ascorbic Acid
Extract
Figure 6: β - Carotene free radical scavenging of Centaurea pullata methanolic extract and
ascorbic acid.
%Scavenging
CPME Scavenging effect against
Hydoxyl radical
3mg/ml
1.5mg/ml
0.75mg/ml 0.37mg/ml
Concentrations
Ascorbic Acid
Extract
Figure 7: Hydroxyl radical scavenging of Centaurea pullata methanolic extract and
ascorbic acid.