Indo European Journal of Scientific Discovery

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2013

Indo European
Journal of Scientific
Discovery

IN-VITRO ANTIBACTERIAL, ANTIFUNGAL, CYTOTOXIC AND INSECTICIDAL

ACTIVITY OF VARIOUS FRACTIONS OBTAINED FROM THE BERRIES OF Berberis
baluchistanica
Faheem Ahmed Siddiqui*1, Mansoor Ahmad2, Mahjabeen,2 Muhammad Imran Sajid1, Muhammad Jamshaid1,
Muhammad Zaman3, Muhammad Nadeem Alvi1, Ali Raza1, Imtiaz Majeed1, Ghulam Jilany Khan1
1

Faculty of Pharmacy, University of Central Punjab, Lahore.

Faculty of Pharmacy, University of Karachi, Karachi.
3
Faculty of Pharmacy, University of Lahore, Lahore.
2

ARTICLE INFO
Keywords
Berberis Baluchistanica
Berries,
Brine Shrimp Cytotoxicity,
Antibacterial Activity,
Antifungal Activity,
Insecticidal Activity.

ABSTRACT
The study was carried out on the berries of plant Berberis baluchistanica Ahrendt.
B.baluchistanica is well known for the cure of immune-deficiency and hepatitis. In this study
the ethanolic extract and butanolic and ethyl acetate fractions of the berries of this plant were
screened for potential antibacterial, antifungal, cytotoxic and insecticidal activities.
Antibacterial Activity was tested using Agar well diffusion method, Antifungal activity was
analyzed using tube dilution method, cytotoxic activity was determined by using brine shrimp
assay and insecticidal activity was measured by paper exposure or impregnation method on
storage pests. Ethyl acetate fraction showed good growth inhibitory activity against Bacillus
cereus (58.31%), Corynebacterium diphtheriae (59.2%), E.coli (30%) and Salmonella typhi
(39.9%). Ethanolic extract of the berries of Berberis baluchistanica showed non-significant
antibacterial activity against tested Gram positive and Gram negative bacteria except
Salmonella typhi. It inhibited 50 % growth of Salmonella typhi as compared to the reference
drug. Mild to moderate antifungal activity was shown by the ethanolic extract against
Trichophyton schoenleinii (23.6%), Pseudoallescheria boydii (50%), Microsporium canis (50
%), Fusarium solani var. lycopersici (15.4%) and Macrophomina phaseolina (28.5%).
However antifungal activity of ethyl acetate and butanolic fractions was found to be nonsignificant. Also, the berries of the plant did not show significant cytotoxic or insecticidal
activity.

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Please cite this article in press as Faheem Ahmed Siddiqui et al. In-Vitro Antibacterial, Antifungal, Cytotoxic and Insecticidal
Activity of Various Fractions Obtained From The Berries of Berberis Baluchistanica. Indo European Journal of Scientific
Discovery. 2013:02(02).

Corresponding author
Faheem Ahmed Siddiqui
Faculty of Pharmacy,
University of Central Punjab, Lahore
+92-3343450671,
+92-42-35880007 Ext 302,
faheem.siddiqui@ucp.edu.pk

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Faheem Ahmed Siddiqui

Vol 2 (2)2015

Copy right 2013 This is an Open Access article distributed under the terms of the Indo European Journal of Scientific Discovery,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
INTRODUCTION
MEDICINAL herbs are gaining increasing importance because of their fewer side effects[1]. It is estimated by the World
Health Organization that 60 to 80 % of the world population still rely on traditional herbal medicine for their health care needs.[2] It is
important to evaluate traditional medicinal herbs for providing the scientific evidence for their proposed effects. It is found that
medicinal plants and herbs contain various chemicals which have potential biological activity[3]. For instance, naturally occurring
essential oils and phenolic compounds possess powerful biological effects[4].
The largest province of Pakistan with respect to area is Balochistan and it is believed this province is rich in plants of
medicinal importance [5-7]. Berberis baluchistanica belongs to Family Berberidaceae; it is popular with the name zarch in local
language Brahvi. It is found abundantly in Kalat district of Balochistan province. This plant is used as fodder for grazing animals
and is also employed as traditional medicine by local people. They use its roots decoction for the cure of internal injuries and
ophthalmic problems[5]. Berberine containing herbs found around the world are primarily used for anti-inflammation.[8]The powder
of its roots and sap is mixed with milk to obtain syrup which is used to heal pain of joints and other kinds of pain in the body such as
pain through injury, rheumatism and chest infection[9]
The first report on its chemical work was reported in 1972, by Shama and coworkers. They isolated two dimeric isoquinoline
alkaloids, Pakistanine and pakistanamine from the roots of the plant[10]. In 1974, again Shama et al. reported the isolation of another
alkaloid Baluchistanamine from the basic extracts of the plant[11]. Since then alkaloids baluchistanine, dihydrosecoquettamine,
secoquettamine, quettamine chloride and gandaramine have been isolated from the plant.[12, 13]. Different parts of the plants have
been evaluated previously for biological activities[5, 7]. In the present study, the alcoholic extract and various fractions obtained from
the berries of Berberis baluchistanica were tested for potential antibacterial, cytotoxic, antifungal and insecticidal activities.
METHODOLOGY
Berries of B. baluchistanica (Ahrendt.) were soaked in ethanol for 20 days. The extract was filtered and dried through rotary
evaporator. The extract was then partitioned between water and ethyl acetate. The ethyl acetate layer was separated and dried under
reduced pressure and at a controlled temperature of 40C. The water layer was then subsequently partitioned with chloroform and presaturated n-butanol. After separating the layers, the dried fractions were obtained by repeating the same process. As a result ethanolic
extract and ethyl acetate and butanolic fractions were collected and subjected to different biological assays. During these assays, the
extract and fractions were screened for antibacterial, cytotoxic (LD 50 against brine shrimps), antifungal and insecticidal activities.

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CYTOTOXIC ACTIVITY
Cytotoxicity of a compound can be checked readily in vivo against brine shrimp larvae[15]. So this bioassay provides a rapid
preliminary screening test for the discovery of compounds with potential toxic activity. Eggs of Artemia salina (brine shrimp) were
stored at low temperature (4). The hatching tray (22 X 32 cm) was half filled with brine solution. 50mg of brine shrimp eggs were
then added. The hatching tray was incubated at 27C for two days in an incubator. The brine shrimp larvae were transferred to well
containing dishes with the help of light and Pasteur pipette. The test sample(20 mg) was used to make 2ml of stock solution from
which 500l, 50l and 5l were added to vials (3 vials for each concentration) corresponding to 1000, 100 and 10g/ml. The solvent
was then allowed to evaporate. After hatching and maturation as nauplii, 10 larvae were taken in each vial. The volume in the vials
was made to 5 ml by adding seawater. The vials were kept at 25-27C for 24 hours under illumination. After 24 hours, the number of
survivors was recorded. The solvent and the reference cytotoxic drug were used as negative and positive control respectively. Finney
Computer program was used to analyze the Data for determining LD 50 value with 95% confidence intervals.

ANTIBACTERIAL ACTIVITY
Agar Well Diffusion Technique[14] was used to determine the antibacterial activity of the various fractions obtained from the
berries of Berberis baluchistanica. In this technique, the wells were made in the media by a metallic borer, which has been sterilized
prior to use. The centers of the wells were at least 24 mm apart. Bacterial inoculum having 102 to 106 CFU/ml (Colony Forming Units)
was spread on the culture medium using sterile cotton swab. The bacterial colony was two to eight hours old. Inside of the well was
cleaned with cotton swab to remove excess fluid. Agar surface of the plate was cleaned with cotton swab three times.
Recommended concentrations of the test sample (2mg/ml of Dimethyl sulfoxide (DMSO) were poured in their respective
wells. Other wells were also provided with DMSO and reference drug, which were used as negative and positive controls,
respectively. The prepared culture plates were then incubated readily at 37C. The incubation time was between 14 to19 hours.
The antibacterial activity was determined by measuring zones of inhibition showing complete inhibition (mm). Growth
inhibitory effect was calculated with reference to positive control.
Ethanolic extract and ethyl acetate and butanolic fractions obtained from the berries of B. Baluchistanica were tested in
concentrations of 200g/100ml of DMSO against the Gram Positive Bacteria including Staphylococcus aureus, Staphylococcus
faecalis, Corynebacterium diphtheria, Bacillus cereus. The Gram Negative Bacteria tested were Escherichia coli, Klebsiella
pneumonia, Proteus mirabilis, Pseudomonas aeruginosa, Salmonella typhi and Shiegella boydii

Faheem Ahmed Siddiqui

Vol 2 (2)2015

ANTIFUNGAL ACTIVITY
Antifungal activity of different fractions obtained from the berries of Berberis baluchistanica (Ahrendt.) was determined by
using Sabourand Dextrose Agar (SDA). Tube dilution method was employed for this purpose[16]. Different fungi used for in-vitro
fungicidal bioassay were Epdermophuton floccosum, Trichophyton schoenleinii, Trichophyton rubrum, Pseudoallescheria boydii,
Candida albicans, Aspergillus niger, which usually infect human beings. Fungi affecting animals used in this experiment included
Microsprum canis, Trichophyton simii and Trichophyton mentagrophytes and finally the extract was also tested against fungi affecting
plants which were Fusarium oxysporum var. lycopersici (tomato), Fusarium solant var. lycopersici (tomato), Macrophomina
phaseolina and Rhizoctonia Solani.
Stock solutions of test sample and crude extract/fractions were prepared by separately dissolving 24mg of extract and 12gm
of pure compound (test sample) in 1ml of DMSO.
SDA medium was prepared by mixing 32.4 grams Sabourand, 4% glucose agar and 4 grams of agar-agar in 500 ml of
distilled water. It was dissolved by seam. 4 ml of it was dispensed into screw cap tubes. These tubes were autoclaved for 15 minutes at
121C. These tubes were then cooled to 50C. Non-solidified agar media was added. From the stock solution 66.6 L of test
compound was added to get final concentrations of 400 and 200g/ml of SDA for crude extract and the pure compound respectively.
In order to solidify, the tubes were kept at room temperature in slanted position. A piece of inoculum was removed from 7-day-old
culture of fungi and added to each tube. Agar surface streak was used for non mycelial growth. DMSO and reference antifungal drugs
(in separate media) were used as negative and positive control respectively. The tubes were kept in incubator for 7 to 10 days at 27 to
29 C.
The growth was determined by measuring linear growth (mm) and growth inhibition was calculated with reference to the
negative control.
INSECTICIDAL ACTIVITY
Various fractions obtained from the berries of Berberis baluchistanica were screened for insecticidal activity by using paper
exposure or impregnation method on storage pests. Common stored grain pests against which the extract and fractions were tested
were Red Flour beetle (Tribolium castaneum), Rice weevil (sitophilus oryzae), Lesser grain borer (Rhyzopertha dominica), Khapra
beetle (Trogoderma granarium) and Pulses beetle (callosobruchus analis).
PREPARATION OF TEST COMPOUND
Five different concentrations of test sample (1571.33g/cm2, 785.66g/cm2, 392.83g/cm2, 196.41g/cm2 and
98.208g/cm2) were prepared from 20%, 10%, 5%, 2.5% and 1.25% stock solution.
REARING TECHNIQUE
The stored grain pests were reared in the laboratory under controlled temperature and humidity so that the insects of uniform
age and size were available for the experiments. Ten pairs of insects were reared in bottles. Each bottle had 250g of breeding media.
Muslin cloth was used to cover the bottle mouths.

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METHODS OF TREATMENT
Insecticidal activity of the extract and fractions was determined by exposing insects to that and test compound. Different
concentrations of each fraction were applied (1ml of each concentration) to small pieces of filter paper. These pieces were then placed
in Petri dishes.
10 insects were kept in each Petri dish. In order to determine solvent effect a batch of 10 insects was treated by solvent only.
One batch of insects was kept to determine the effects of the environment. 3 rd was used to test the effects of the reference drug which
was Coopex (synthetic Pyrethroid). All Petri dishes were kept for 24 hours after which mortality counts were made. Probit mortality
curve was used to calculate LD50 values [17].

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RESULTS
Table 1. Antibacterial activity of extract and various fractions obtained by berries of Berberis baluchistanica.
Zone of inhibition in mm
Ethanolic extract Butanolic fraction
1
Bacillus cereus
Non-significant
Non-significant
2
Corynebacterium diphtheria Non-significant
Non-significant
3
Staphylococcus aureus
Non-significant
Non-significant
4
Staphylococcus faecalis
Non-significant
Non-significant
Zone of inhibition in mm
Sr # Gram negative bacteria
Ethanolic extract Butanolic fraction
1
Escherichia Coli
Non-significant
Non-significant
2
Klebsiella pneumonia
Non-significant
Non-significant
3
Proteus Mirabilis
Non-significant
Non-significant
4
Pseudomonas Aeruginosa
Non-significant
Non-significant
5
Salmonella Typhi
7.5(50%)
Non-significant
6
Shiegella boydii
Non-significant
Non-significant
(t= decrease in bacterial population/unit area)
Reference drug= Amoxicillin trihydrate
Sr #

Gram positive bacteria

Ethyl acetate fraction

7(58.1%)
8(59%)
Non-significant
Non-significant

Reference drug
12
14
13
20

Ethyl acetate fraction

6(30%)
Non-significant
Non-significant
Non-significant
6(40%)
Non-significant

reference drug
20
16
12
8
15
18

Ethanolic extract of the berries of B. Baluchistanica exhibited a moderate activity (50%) against Salmonella typhi, remaining
gram negative and all gram positive bacteria showed normal growth in the presence of the extract as shown in Table 1. The ethyl
acetate fraction showed good antibacterial activity against strains of Bacillus cereus (58.1%) and Corynebacterium diphtheria (59%),
while week activity was observed against Gram negative Escherichia coli (30%) and Salmonella typhi (40%) strains. The extract was
found to be harmless for other Gram positive and Gram negative bacteria used in the screening process (as shown in table 1).T he
butanolic fraction didnt show any antibacterial activity except slight activity against Gram negative Salmonella typhi(7.5%).
Table 2. In-vitro Cytotoxic activity for the berries of Berberis baluchistanica Ahrendt.
Dose
No. of No.
of LD50
Reference LD50
(g/ml) shrimps survivors (g/ml) drug
(g/ml)
1000
30
30
Ethanolic extract 100
30
30
10
30
30
1000
30
30
Artemia
Copper
Salina(Brine Butanolic fraction 100
30
30
Sulphate( shrimp)
CuSO4)
10
30
30
1000
30
30
Ethyl
Acetate
30
30
30
fraction
10
30
30
None of the extract/fractions of the berries of B. baluchistanica possessed cytotoxic activity.

Extract/Fraction

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Organism

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Table 3. Antifungal Activity of the berries of Berberis baluchistanica.

Fungi

Antifungal activity
Ethanolic extract
Human pathogens

Trichophyton schoenleinii

23.60%

Pseudallescherchia boydii

50%

Candida albicans

0%

Aspergillus niger

Microsporum canis

0%
Animal pathogens
50%

1
2
3

Fusarium oxysporum var. lycopersici(tomato)

Fusarium solani var. lycopersici (tomato)
Macrophomina phaseolina

Plant pathogens
0%
15.40%
28.50%

antifungal activity of Reference drug

reference drug
activity
Miconazole
Ketoconazole
Miconazole
Ketoconazole
Miconazole
Ketoconazole
Amphotericin-P

95%
95%
95%
90%
90%
90%
100%

Miconazole
Ketoconazole

100%
100%

Benlate
Benlate
Benlate

100%
100%
100%

The ethanolic extract of the berries of Berberis baluchistanica was screened for antifungal activity. It showed moderate
activity against Pseudoescheria boydii (a human pathogen) and Microsporum canis (animal pathogen) as is evident from Table 3.
Table 4. Insecticidal test of the berries of Berberis baluchistanica.
Extract/Fraction
Ethanolic extract
Butanolic fraction
Ethyl acetate fraction

Dose in g/cm2
1571.33
1571.33
1571.33

Mortality mean %
0%
0%
0%
0%
0%
0%
0%
0%
0%

The ethanolic extract, butanolic and ethyl acetate fractions obtained from the berries of Berberis baluchistanica were screened
for insecticidal activity. None of the extract and fractions showed any activity against the test organisms (Table 4).

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DISCUSSION
The ethanolic extract of the berries of Berberis baluchistanica showed non-significant antibacterial activity against all Gram
positive and Gram negative bacteria except Salmonella typhi. It inhibited 50 % growth of Salmonella typhi as compared to reference
antibacterial drug amoxicillin trihydrate. The butanolic fractions of berries of Berberis baluchistanica exhibited non-significant results
in both Gram positive and Gram negative bacteria which also indicated the non-toxicity of the fraction. The ethyl acetate fraction
showed growth inhibitory activity in Bacillus cereus (58.31%), Corynebacterium diphtheriae (59.2%), E.coli (30%) and Salmonella
typhi (39.9%). Oleanolic acid was obtained from this fraction and the reported activities of oleanolic acid are antiulcer, antiinflammatory and ant allergic. Due to moderate antibacterial activity of ethyl acetate fraction, it can be assumed this inhibitory activity
might belong to oleanolic acid and its evidential proof needs further research.
The antifungal activity of crude extracts and fractions that is ethanolic extract, butanolic and ethyl acetate fractions were
found non-significant. In ethanolic extract, mild and moderate activity was noticed only in Trichophyton schoenleinii (23.6%),
Pseudoallescheria boydii (50%), Microsporium canis (50 %), Fusarium solani var. lycopersici (15.4%) and Macrophomina
phaseolina (28.5%). However, butanolic and ethyl acetate fractions showed no activity.
The cytotoxic activity was assessed by brine shrimp assay and no cytotoxic activity was observed in the extracts and fractions
obtained from the berries of the plant. Similarly, the extract and fractions did not possess any insecticidal activity. It is important to
note here that in the previous studies conducted on roots of the plant showed significant antileishmanial, cytotoxic, antioxidant
activities.[5, 7] This suggests that the berries of plant vary remarkably in composition to that of roots.

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CONCLUSION
It can be concluded from the present study that the berries of Berberis baluchistanica does not possess significant
antibacterial, antifungal, cytotoxic and insecticidal activity. However, the extracts and fractions obtained from roots and leaves of the
plants have been reported for different biological activities. Further research should be carried out on all parts of the plant to confirm
the biological activities possessed by this medicinal plant.
ACKNOWLEDGEMENTS
We wish to express our thanks to Faculty of Pharmacy, University of Karachi, Pakistan for providing facilities in term of
equipment and technical assistance. We would like to extend our deep and sincere gratitude to Professor Dr. Ghazala Hafeez, Dean
Faculty of Pharmacy for her encouragement and support. Furthermore, we would like to acknowledge the Faculty of Pharmacy,
University of Central Punjab for encouragement in research activities.

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