SYNERGISTIC ANTIBACTERIAL STUDIES OF HELIOTROPIUM STERIGOSUM
Tahira Mughal1, Ismat Naeem2, Sadia Qureshi 3, Ammara Abass2
1. Department of Botany, Lahore College for Women University, Jail Road, Lahore, Pakistan 2. Department of Chemistry, Lahore College for Women University, Jail Road, Lahore Pakistan 3. Department of Biochemistry, Avicenna Medical College, Bedian Road Lahore, Pakistan
Keywords: Heliotropium sterigosum is an annual herb found in south Punjab of Pakistan. Extract of whole plant in methanol was tested for anti bacterial activity against Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Streptococcus pneumoniae, Bacillus subtilus, and Sarcina lutae. Minimum Inhibitory Concentration of methanolic extract was determined and found to be bactericidal in concentration of 1000 µg/ml against streptococcus pneumoniae, Escherichia coli, and Streptococcus pneumoniae. Synergistic antibacterial activity of methanolic extracts was tested with respective solvent extracts of seeds of Withania coagulans, whole plant of Hypericum perforatum and Pinus roxburgii (bark). Staphylococcus aureus was synergistically inhibited by methanolic extract of Heliotropium sterigosum and Pinus roxburgii (bark) (1µg/ml) and Withania coagulans (1µg/ml). Methanolic extract of Heliotropium sterigosum inhibits the growth of Sarcina lutae in combination with Pinus roxburgii (bark) methanolic extract (0.5µg/ml). Highest synergistic activity was observed by Heliotropium sterigosum and Withania coagulans methanolic extracts against Pseudomonas aeruginosa (0.5µg/ml) and Bacillus subtilus (1µg/ml).
Abstract

Heliotropium sterigosum, antibacterial activity, synergistic activity, Withania coagulans, Pinus roxburgii and Hypericum perforatum.

Article Information

Identifiers and Pagination:
Year:2010
Volume:2
First Page:27
Last Page:36
Publisher Id:JAppPharm (2010 ). 2. 27-36
Article History:
Received:November 13, 2009
Accepted:January 5, 2010
Collection year:2009
First Published:January 13, 2010

INTRODUCTION
The medicinal plants have applications in pharmaceutical, cosmetic, agricultural and food industry. The use
of medicinal herbs for curing diseases has been documented in history of all civilizations. Man in the
prehistoric era was probably not aware of the health hazards associated with irrational therapy. With
advancement in the field of therapeutics, it was concluded that plants contains active principles, which are
responsible for curative action of the herbs. Ritch-Krc, et al. reported biological activity of some pinus
species by pitch preparations against known human pathogens e.g.; Escherichia coli, Staphylococcus
aureus, Pseudomonas aeruginosa, Candida albicans and Aspergillus fumigatus [Ritch-Krc,et al.2005]
Antibacterial properties of aqueous and methanolic extracts of 26 medicinal plants used in Mexico to treat
gastrointestinal disorders were tested against eight different species of enteropathogens: two Escherichia
coli species, two Shigella sonnei species, two Shigella flexneri species, and two Salmonella sp. species
[Alanís et al., 2005].
Antibacterial activity of Ashwagandha [Withania somnifera L. Dunal (Solanaceae; root and leaves)], an
Indian traditional medicinal plant has been reported against pathogenic bacteria. Oral administration of the
aqueous extracts successfully obliterated salmonella infection in Balb/C mice as revealed by increased
survival rate as well as less bacterial load in various vital organs of the treated animals. [Owais, et al.,
2005]
The genus Heliotropium sterigosum, an annual herb consists of about 10-15 species and belongs to family
Boraginaceae. It is widely distributed in the South Punjab specially Sahiwal to Bahwalpure road Pakistan.
Few species have also been reported from Lahore. Ethno medicinally the plant is laxative and diuretic. The
juice is used as an application to sore eyes, gum boils and sores. The decoction of these plants is used as a
cure for pain in the limbs [Kiritikar and Basu 1987]
The alkaloid europine isolated from Heliotropium bovei showed both antifungal and insect antifeedant
activity [Reina M, et al, 1995]. Individual alkaloids (lasicarpine, heliotrene, supinine and 9-
angeloylretronecine N-oxide) were isolated from H. burseriferum. [Marquinca G.A, 1989].
Alcoholic extract of H. indicum was found to possess dose-dependent anti microbial activity against all the
test organism [Rao PR.S Nammi and ADV Raju, 2002].
The increase failure of chemotherapeutics against anti biotic resistant exhibited by pathogenic microbial
infectious agent, has led to the screening of several medicinal plants for their potential antimicrobial
activity [Martinus, et.al, 2001] [Scazzocchio, et al, 2001].
In search of the antibiotics and protection against infectious disease widespread in Pakistan, Heliotropium
sterigosum (whole plant) was selected. Its antibacterial activity and synergistic activity with other herbs
i.e.; Pinus roxburgii (bark), Withania coagulans and Hypericum perforatum whose antibacterial activity
has already reported was checked [Naeem I, and Maimoona A, 2008] [Naeem I, et al, 2007] [Siddique Z,
2008].


MATERIAL METHOD
Microbial strains of Staphylococcus aureus (ATCC 25923) Escherichia coli (ATCC 2592), pseudomonas
aeruginosa (ATCC 27853) Streptococcus pneumoniae (ATCC 49619) Bacillus subtilus (ATCC 6051) and
Sarcina lutae (ATCC 9341) were obtained from National institute of Health Islamabad.
Plant material
Fresh plants of Heliotropium sterigosum (whole plant (PM # 040), Withania coagulans (seeds)(PM # 004)
were collected from South Punjab (Bahawalpur Road / Sakhi Sarwar ) on 6th June 2005 and Hypericum
perforatum (whole plant, PM # 0131) and that of Pinus roxburgii (bark) (PM # 0157) from area between
Nathia Gali, Khanuspure and Muree Hill, Pakistan on 2nd October 2005. The plants were identified by Mir
Ajab Ali Khan, Professor of Botany Quide-e-Azam university , Islamabad, Pakistan, Dr. Zaheer-ud-din ,
Professor of Botany Government College Lahore, Pakistan and voucher specimen deposit in Prem Madan
Herbarium of Lahore College for Women University, Lahore, Pakistan.
Heliotropium sterigosum (whole Plant), Withania coagulans (seeds) Hypericum perforatum (whole plant)
and Pinus roxburgii (bark) were air dried and extract was obtained by soxhelt extraction to yield 15%,
20%, 18% and 25% solvent free extract.
Anti-bacterial activity was determined by agar well diffusion method [Norsel and Messley, 1977]. This test
was performed in triplicate by spreading 12-18 hour old pathogenic bacterial cultures containing
approximately 106 - 1010 colony forming unit (CFU/ml). On the surface of nutrient agar plates, wells
(4mm) were dug in the media with the help of sterile metallic borer.
Test samples of different concentrations prepared in Methanol were added (50µl) in their respective wells
pure methanol was used as negative control (3mm). Other wells were supplemented with reference
compounds i.e. Ampicillin, Amoxicillin, Levofloxin, Tetracycline, Vancomycin, Ciprofloxacin and
Penicillin as positive control.
Synergistic Activity against bacterial strains was determined by taking equal amount (50µl (1:1) of plant
extracts by agar well diffusion method.


RESULTS AND DISCUSSION
Six pathogenic bacterial strains (Staphylococcus. aureus, Escherichia. coli , Bacillus subtilus
Streptococcus pneumoniae ,Sarcina lutae and Pseudomonas aeruginosa were used and methanolic extract
of one plant (H.sterigosum) with three combinations of methanolic extracts of other plants
(H.sterigosum+Hypericum Perforatum, H.sterigosum+Pinus bark (rox) and H.sterigosum+W.coagulans)
with concentration (1000,250,100,50,10,5 and1µg/ml.) were used against each of the six bacterial strains.
From the results obtained it was observed that methanol was the best solvent for extracting antimicrobial
substances from tested medicinal Plant based on the number of organisms inhibited and the diameter of
inhibitory zones produced. It was also observed that different extracts were different in their antimicrobial
effectiveness depending on the extractive solvent used. Our results prove favorably the suggestion of Oloke
and Kolawole [Oloke and Kolawole, 1988] that bioactive components of any medicinal plant may differ in
their solubility depending on the extractive solvents used. The crude methanolic extract of H.sterigosum
showed highest bactericidal activity against S.aureus, P.aurginosa, S. pneumoniae and E.coli. The crude
methanolic extract of H.sterigosum showed poor antibacterial activity against B. subtilus and S.lutea
(Table 1 and 3)
The Withania coagulans seed crude methanolic extract showed good antibacterial activity against S.aureus,
B.subtilus but were moderately active against E. coli. P. aeruginosa, (Table 1 and 4)
The methanolic extract of Pinus roxburghii (bark) showed highest antibacterial activity against the
bacterial strain S.aureus, P.aeruginosa and E.coli. Their MIC value ranges from 0.1µg/µl-0.5µg/µl. (Table
1 and 5)
The Hypericum perforatum showed highest activity against S.aureus, P. aeruginosa and E.coli. The MIC
value of the plant ranges from 0.1µg/µl-0.5µg/l. (Table 1and 6)
Synergistic activity: As the antibacterial activity of methanol extract was better than all other solvent
extracts it was selected to study synergistic effect in combination with respective extract of Heliotropium
sterigosum with Hypericum perforatum, Withania coagulans, Pinus roxburgii (bark). This combination
suggested increase in the inhibitory effect of crude drug extract against different microbes that may or not
have developed resistant to many available antibiotics.
Synergistic activity (inhibition is doubled compared to one of the constituent ) was observed in as low in
amounts as 0.1ug /ml (100ug/ml )methanolic extract of Heliotropium sterigosum mixed with Withania
coagulans.The growth of Streptococcus pneumoniae and E.coli was inhibited completely . (Table 7and 8)
Heliotropium sterigosum also showed synergistic activity with Pinus roxburgii (bark) against Escherichia
coli, Staphylococcus aureus and Streptococcus pneumoniae (Table 7 and10). The growth of all bacteria was
inhibited when methanolic extracts of Heliotropium sterigosum and Hypericum perforatum were mixed.
The growth of all tested bacterial growth was asserted by mixing the methanolic extracts of Heliotropium
sterigosum and Hypericum perforatum. (Table 7 and 9)

Table – 07 MIC value of crude methanol extracts of Heliotropium sterigosum with other (Pinus
roxburgii (bark) Withania coagulans, Hypericum perforatum) medicinal plants

Table -08 Zone of inhibition of crude methanol extracts of Heliotropium sterigosum and Withania
coagulans

Table – 09 Zone of inhibition of crude methanol extracts of Heliotropium sterigosum and Hypericum
perforatum

Table -10 Zone of inhibition of crude methanol extracts of Heliotropium sterigosum and Pinus
roxburgii (bark)


CONCLUSION
It is concluded that the methanolic extract of Heliotropium sterigosum has antibacterial activity and has
maximum effects against Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli.
Moreover, it also has synergistic effect when mixed with Withania coagulans, Pinus roxburgii (bark) and
Hypericum perforatum.


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Book Distributors , Dehra Dun , India.


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Welcome to the research group of Prof. Dr. Cornelia M. Keck in Marburg. Cornelia M. Keck is a pharmacist and obtained her PhD in 2006 from the Freie Universität (FU) in Berlin. In 2009 she was appointed as Adjunct Professor for Pharmaceutical and Nutritional Nanotechnology at the University Putra Malaysia (UPM) and in 2011 she obtained her Venia legendi (Habilitation) at the Freie Universität Berlin and was appointed as a Professor for Pharmacology and Pharmaceutics at the University of Applied Sciences Kaiserslautern. Since 2016 she is Professor of Pharmaceutics and Biopharmaceutics at the Philipps-Universität Marburg. Her field of research is the development and characterization of innovative nanocarriers for improved delivery of poorly soluble actives for healthcare and cosmetics. Prof. Keck is executive board member of the German Association of Nanotechnology (Deutscher Verband Nanotechnologie), Vize-chairman of the unit „Dermocosmetics“ at the German Society of Dermopharmacy, active member in many pharmaceutical societies and member of the BfR Committee for Cosmetics at the Federal Institute for Risk Assessment (BfR).

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doi: http://dx.doi.org/10.21065/19204159
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