International Journal of Indigenous Medicinal Plants, ISSN: 2051-4263, Vol.46, Issue.2
1217
Biological Screening of Methanolic Crude Extracts
of Caralluma Tuberculata
Muhammad Zahid Khan
Department of Biotechnology, Faculty of Biological Sciences, University of Science and Technology Bannu, KPK,
Pakistan
Rahmat Ali Khan
Department of Biotechnology, Faculty of Biological Sciences, University of Science and Technology Bannu, KPK,
Pakistan
Mushtaq Ahmed
Department of Biotechnology, Faculty of Biological Sciences, University of Science and Technology Bannu, KPK,
Pakistan
Nawshad Muhammad
Department of Biotechnology, Faculty of Biological Sciences, University of Science and Technology Bannu, KPK,
Pakistan
Muhammad Rashid Khan
Department of Biochemistry, Faculty of Biological Sciences, Quiad-i-Azam University Islamabad, Pakistan
Hidayat Ullah Khan
Department of Biotechnology, Faculty of Biological Sciences, University of Science and Technology Bannu, KPK,
Pakistan
Nagina Atlas
Department of Biotechnology International Islamic University; Faculty of Basic and Applied Sciences, Islamabad,
Pakistan
Farman Ullah Khan
Department of Chemistry, University of Science and Technology Bannu, KPK, Pakistan
Corresponding author Email: rahmatgul_81@yahoo.com
ABSTRACT
Caralluma belongs to the family Asclepiadaceae, has
about hundred species, dispersed in various countries
which includes Spain, Saudi Arabia, Africa, Middle East,
India, and Pakistan. Caralluma tuberculata is
traditionally used in treatment of various human ailments
in Pakistan. Presently we arranged to investigate the
antioxidant, antimicrobial, phytoxic and cytotoxic
activities. DPPH (1, 1-diphenyle -2- picryle hydrazyl) free
radical scavenging activity revealed that CTME is very
potent as compare to control. CTME also showed
significant activity in microbial inhibition as well as
phytoxic and cytotoxic activities.
Key words- Caralluma tuberculata, DPPH, Phytoxicity;
Cytotoxicity; Asclepiadaceae
1. INTRODUCTION
Plants have the capability an extensive diversity of
chemical compounds that are used to carry out vital natural
functions, and to protect against attack from predators. On
long-term many of these phytochemical have valuable
effects when consumed by humans, and their usage is
effective in the treatment of various diseases. So far at
slightest 12,000 such compounds have been isolated; a
number predictable to be less than 10% of the total [1-2].
Caralluma belongs to the family Asclepiadaceae, has about
hundred species, dispersed in
various countries which includes Spain, Saudi Arabia,
Africa, Middle East, India, and Pakistan. In Pakistan, two
species of Caralluma is found, C. edulis and C. tuberculata
[3]. Caralluma has dominant medicinal importance found
in the dry regions of the world and possess antiinflammatory and anti-tumor activity [4-6]. Due to the
presence of the pregnane glycosides in Caralluma it
possesses anti-tumor and anti-cancer properties [7, 8].
Caralluma tuberculata juicy stem is sour tonic,
carminative, febrifuge, and stomachic valuable in
rheumatism and consumed as vegetable [9]. Traditionally
in Pakistan both urban and rural population, used caralluma
as an anti-diabetic therapeutic agent [11]. In semi arid areas
of Pakistan Caralluma species have been used for centuries
as emergency foods [12-13] and other Caralluma species
for their anti-hyperglycemic activity [14] and joints pain
[15].
2. MATERIAL AND METHODS
2.1 Plant Preparation and Extraction
A fresh Caralluma tuberculata whole plant was
purchased from local market district Bannu, Pakistan,
identified by Prof. Abdur Rahman, Department of
Botany, Post graduate college Bannu and submitted in
the Herbarium, University of Science and Technology
Bannu. Plant was chopped, kept under shadow for
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1218
dryness and converted into fine powder with the help
of mechanical grinder. 800 g fine powder was socked
in 2 liter of 80 % methanol for seven days at room
temperature and filtered. The filtrate was collected, and
evaporated under reduced pressure in rotary
evaporator. The crude extract was store at 4 C o for
further analysis.
2.6 Hatching of shrimps
2.2 Phytotoxic Assay
2.7 Bioassay
In this petri plate studies the protocol of Khan et al. [1621]; and Ahmed et al. [22].
Drum vials were used in this bioassay. 0.5 ml of each
solution (1000 μg/ml, 100 μg/ml, 10 μg/ml) was taken in
vials and evaporated the solvents. Residues were resolved
in saline of 2 ml. To every vial 10 shrimps were placed and
raised the volume up to 5 ml and incubate at 25–28 °C,
following 24 hr of incubation survivors were measured with
help of 3× magnifying glass and calculation was done using
Abbot’s formula;
2.3 Antioxidant Assay
The Gymfi et al., [23] procedure with some modifications
was followed for this assay of DPPH (1, 1-diphenyle -2picryle hydrazyl).
2.4 Antifungal assay
Brine shrimps were hatched in two compartment
rectangular tray containing sea salt saline. Eggs were
sprinkled in dark compartment of tray and after 24 hr of
shrimps hatching larvae was collected by pipette from the
lightened side.
% Death = (Sample-control/control) × 100.
For characterization of antifungal activities of the methanol
extract of the Caralluma tuberculata the standard protocols
of Duraipandiyan and Lgnacimuthu [24]; Ahmed et al [25]
was followed.
2.5 Cytotoxic brine shrimp assay
2.5.1 Saline preparation
28 g sea commercial sea salt (sigma) was dissolved in one
liter of dH2O with continuous stirring for 2 hr.
3rd Day
3. RESULTS
3.1 Phytotoxic activity
To study the phytotoxic activity of the Caralluma
tuberculata methanolic extract (CTME), 1000µg/ml
concentration of the sample was used. The obtained results
show that CTME inhibits the growth of shoot and roots
(hypocotyls & radicals) of the rice seeds (Oryza sitiva) as
compared to the control, as shown in the Figure 1-2.
7th Day
10th Day
Figure.1. Shoot growth of rice in the presence (1000µg/ml) and absence of CTME
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3rd Day
7th Day
1219
10th Day
Figure 2. Roots growth of rice in the presence (1000µg/ml) and absence of CTME
3.2 DPPH free radicals scavenging activity
DPPH free radicals have the ability to take electrons from
the anti-oxidants that is why it is used for the in vitro
antioxidants scavenging assays of the medicinal plants for
its estimation. Table1 and Figure 3 shows % scavenging
activity of CTME vs ascorbic acid. By using the CTME
with the increasing concentration, positive scavenging
activities were observed and it was noted that the
scavenging
activity
of
50µg/ml<100µg/ml<150µg/ml<200µg/ml<250µg/ml<500
µg/ml which shows their similarities to the ascorbic acid
scavenging activities which were used as a reference.
Table1. Comparison b/w CTME and ascorbic acid scavenging activity
Concentration (in µg/ml)
% CTME Scavenging
% Ascorbic acid Scavenging
50
33
18.2
100
36.5
20.38
150
40.8
30.28
200
250
500
44.12
50.15
75.2
35.7
40.9
60.3
75.2
60.3
36.5
33
25.38
40.8
30.28
44.12
35.7
50.15
40.9
18.2
50µg /ml
100µg /ml 150µg/ml 200µg/ml 250µg/ml 500µg/ml
Figure3. Antioxidant activity of CTME
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International Journal of Indigenous Medicinal Plants, ISSN: 2051-4263, Vol.46, Issue.2
3.3 Antifungal Activity
For the screening of anti-fungal activity of the sample; 67
µl (200µg/ml) of the CTME, 67 µl (200µg/ml) of the
terbinofine and 67 µl of the DMSO (99.9 %) were used.
CTME anti-fungal activity to some extant against
1220
Aspergillus niger and Aspergillus flavius. The DMSO
shows zero percent inhibition against these used fungal
strains, and the terbinofine was indicated highly active
against these strains i.e. shows great inhibition to these
fungal growths as in the Table 2.
Table.2. Antifungal activity of Caralluma tuberculata methanolic extracts (CTME):
T. No
Niger growth
on DMSO
Niger growth
on
TB
Niger
growth on
CTME
Flavius growth
on DMSO
Flavius
growth on
TB
Flavius
growth on
CTME
1
8.8 cm
3.5 cm
4.2 cm
8.8 cm
0.0 cm
2.8 cm
2
7.5 cm
5.5 cm
3.8 cm
8.5 cm
0.0 cm
2.3 cm
Average growth
8.15 cm
4.5 cm
4.05 cm
8.65 cm
0.0 cm
2.55 cm
%Inhibition
0.00 %
44.78%
50.30 %
0 .00 %
100 %
71.52 %
3.4 Cytotoxic brine shrimp assay
CTME shows cytotoxic activity the results are given in the
Table No 3 and Figure No 4. CTME possesses excellent
cytotoxic activity at 1000 ug/ml of which is 100%
activity ,at the (100 ug/ml) concentration the plant extract
also shows 100% cytoxicity activity while at (10 ug/ml). It
shows 70% cytotoxic activity. The cytotoxic activity in
control shows no inhibition.
Table3. Cytotoxic activity of CTME
Conc (µg/ml)
No.of subjects
No.of living
No.of death
Control
10
10
0
100%
10 µg/ml
10
7
3
70%
100 µg/ml
10
0
10
100%
10
0
10
100%
1000 µg/ml
Percentage activity (%)
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1221
Figure4. Cytotoxic activity of CTME:
Plant Extract
10
%Death
8
5
3
0
Control
10 µg/ml
100 µg/ml
Concentration
1000 µg/ml
Figure4. Cytotoxic activity of CTME:
4. DISCUSSIONS
Medicinal plants play its fundamental function for the
treatment of diverse human aliments due to the presence of
bioactive compounds in them, such as the treatment of
inflammation, oxidative stress, heart diseases and cancer
etc. Because of their less side effects as compare to the
synthetic drugs different parts of the medicinal plants
(natural products) are used generally for the treatment of a
variety of diseases. In the world Local herbal system is
extensively used. In a similar way Pakistan is also a
prosperous country in medicinal plants which are locally
used in folk medicines for the treatments of various diseases
like infections, cardiovascular diseases, diseases of
digestive system and skin diseases etc by local healers.
Therefore, the basis of current study was to investigate the
pharmacological status of caralluma tuberculata plant.
Phytotoxicity of medicinal plants are also very significant
because of its growth inhibition of weeds and other
unwanted plants. The phytotoxic results obtained from
CTME showed that CTME inhibit the growth / germination
of roots and shoots of rice (Oryza sitiva) as compare to the
other medicinal plants methanolic extracts, such as
significant phytotoxic results were found by Kordali et al.,
[26], reported that essential oils and phenolic compounds
inhibits the growth of roots and shoots. In the every living
systems for the production of energy “oxidation” is one of
the significant and essential processes, however in this
oxidation process (oxygen consumption during normal
metabolism) the RFR (reactive free radicals) are formed by
diverse enzymatic systems. The ROS (reactive oxygen
species) in minute amounts are valuable for growth
regulation and signal transduction, but ROS in huge
amounts generate oxidative stress which attacks and harm
various molecules like DNA, Protein and Lipids [27-33].
The obtained data from the present study of Caralluma
tuberculata methanolic extracts (CTME) show significant
scavenging potential. The anti-oxidant potential of the
methanolic extracts of the medicinal plants are due to the
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International Journal of Indigenous Medicinal Plants, ISSN: 2051-4263, Vol.46, Issue.2
phenolic and polyphenolic compounds present in them,
which noticeably reduce the free radicals that cause the
oxidative stress (damage the molecules). Our results show
some similarities with the
investigation [34-37],
reported that free radicals are markedly scavenge by
medicinal plants. As fungi produce mycotoxin which
considerably effect human health, some fungus also destroy
our foods and grains; disturb their nutritional value thus
made them unhealthy for our use [37-40]. In our present
study of Anti-fungal activity of (CTME), the obtained result
show that fungal strains are significantly inhibited.
Brine shrimp lethality is a common bioassay, which is
investigative of antibacterial activities cytotoxicity,
pesticide effects and diverse pharmacologic activities [41].
The results point out the capability of the plant extract in
cell cultures to kill cancer cells, eradicate pests, and
exercise a broad choice of pharmacological activities [42].
In our present study of cytotoxic activity of CTME, the
obtained result indicates that Caralluma tuberculata
methanolic extract possess cytotoxic potential.
5. CONCLUSION
From the data it is recommended that Caralluma
tuberculata methnolic extract should be used as a
potent antioxidant, pytotoxic and antifungal and cytotoxic
agent.
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