Ethnobotanical Leaflets 14: 445-56, 2010.
Screening for Antibacterial Activity of Andrographis paniculata Used in Malaysian Folkloric Medicine: A Possible Alternative for the Treatment of Skin Infections
A. Sule1, Q.U. Ahmed*2, O.A. Samah1, and M.N. Omar1
1Department of Biomedical Sciences, Faculty of Science
2Department of Pharmaceutical Chemistry, Faculty of Pharmacy
*Corresponding Author: Email: firstname.lastname@example.org
Issued: April 01, 2010
In this study non-polar (dichloromethane) and polar (MeOH & aqueous) extracts of A. paniculata (whole plant) were evaluated for in vitro antibacterial activity against 12 skin disease causing bacterial strains (7 gram positive� strains; Staphylococcus saprophyticus, Staphylococcus epidermis, Staphylococcus aureus, Streptococcus pyogenes, Bacillus anthracis, Micrococcus luteus, Enterococcus faecalis) and 5 gram negative strains; Proteus mirabilis, Proteus vulgaris, Klebsiella pneumoniae, Neisseria meningitis, Pseudomonas aeruginosa ) using the disc diffusion method at three concentrations; 1000, 500, and 250 �g/disc respectively in order to ascertain its folkloric claim to treat skin infections. The extracts showed significant antibacterial activities against both the Gram-positive and Gram-negative bacterial strains tested. Highest significant antibacterial activity was exerted by the MeOH extract against E. faecalis at 1000 �g/disc (24.00 � 0.00 mm) and the least activity by the DCM extract against N. meningitis at 250 �g/disc (6.00 � 0.00mm). The minimum inhibitory concentration ranged between 150 �g /mL and 300 �g /mL depending on microorganism and various extracts. Presence of phytochemicals such as terpenoids, tannins, flavonoids, saponins, alkaloids, amino acids and steroids were observed. These results candidly suggest the presence of promising antibacterial substances in the polar as well as non-polar extracts which could be potential phytomedicine for the treatment of skin infections caused by pathogenic bacterial strains. These findings explicitly support its traditional claims and form a strong basis for further efforts to explore A. paniculata�s antibacterial potential to treat skin frailties efficaciously. Our results confer the utility of this plant extracts in developing a novel broad spectrum antimicrobial agent.
Keywords: Andrographis paniculata, Acanthaceae, antimicrobial activity, skin infections, MIC.
����������� There have been high rise in the frequency of certain
skin infections in developing countries including
paniculata (Burm.f.) Wall. ex Nees., also
known commonly as "King of Bitters (English) or Hempedu Bumi
(Malay)," is a member of the plant family Acanthaceae. It is an annual herbaceous plant which is widely
cultivated in southern Asia,
����������� A. paniculata has been used in the treatment of some skin infections in
Materials and Methods
Collection and preparation of plant material
plant material (5kg) of Andrographis
paniculata was procured from the botanical gardens of the Forest Research
Institute of Malaysia (FRIM),
Preparation of non-polar and polar extracts
����������� 500g dry powder of A. paniculata (whole plant) was sequentially extracted with dichloromethane and methanol using the Soxhlet apparatus on the water bath for 12 h each (Harborne, 1998). Each of the mixtures was carefully filtered using filter paper (Whatman No. A-3) and concentrated using a rotary evaporator (Buchi Rotary Evaporator, R-210) at 400C. The final concentrated extracts were stored at -18�C in labeled sterile bottles and kept as aliquots until further evaluation.
����������� Another 500 g of powdered sample of the herb was extracted by soaking in 1 L double distilled water in a round bottom flask, stirred for about 6 min, closed tight using a rubber cork and left overnight at room temperature. Thereafter, the solution was filtered using filter paper (Whatman No. A-1) and extract was freeze dried and carefully stored at -18�C in labeled sterile bottles.
����������� Twelve skin disease causing bacterial strains were
taken into consideration, viz., (7 Gram-positive: Staphylococcus saprophyticus-IMR S-1242, Staphylococcus epidermis-IMR S-947, Staphylococcus aureus-IMR S-277, Streptococcus pyogenes-IMR S-526, Bacillus anthracis-IMR B-132, Micrococcus luteus-IMR B-7, Enterococcus faecalis-IMR E-150 and 5 gram
negative strains: Proteus mirabilis-IMR P-76, Proteus vulgaris-IMR P-147, Klebsiella pneumonia-IMR K-6, Neisseria
P-84. All bacterial strains were purchased directly from the
Institute for Medical Research (IMR),
Screening for Antibacterial Activity
����������� The agar disc diffusion method was employed for the
determination of antibacterial activities of the extracts of A. paniculata (NCCLS, 2004). 7
gram-positive (S. saprophyticus, S.
epidermis, S. aureus, S.
pyogenes, B. anthracis, M.
luteus, E. faecalis)
and 5 gram-negative (P. mirabilis, P. vulgaris, K. pneumoniae, N. meningitis, P. aeruginosa) standard bacterial strains of human skin disorders were used.
All bacterial cultures were first grown on nutrient agar plates at 37 oC
for 24 h. Few colonies (2 to 3) of similar morphology of the respective
bacteria were transferred to a liquid medium (Mueller Hinton Broth) and
incubated until adequate growth of turbidity equivalent to McFarland 0.5
turbidity standard was obtained. The inocula of the respective bacteria were
streaked on to the Mueller Hinton plates. The dried plant extracts were
dissolved in 10% aqueous dimethyl sulfoxide (DMSO) and sterilized by
filtration through a 0.45 mm membrane filter. Sterile filter paper discs (5
mm) (Whatman no. 1) were punched and impregnated with 10 l of the DCM,
MeOH and aqueous extracts (corresponding to 1000, 500, and 250 �g/disc) and
allowed to dry at room temperature. These were placed on the Mueller-Hinton
agar plates inoculated with the test strains. The plates were then allowed to
stay for 1 h at room temperature and finally incubated at 37 oC
for 24 h (Heraeus GmbH, D-6450, and
Determination of minimum inhibitory concentration (MIC)
����������� The minimum inhibitory concentration (MIC) of the crude
extracts of A. paniculata was
determined by agar dilution method (EUCAST, 2000). The growth media,
Mueller-Hinton agar (MHA) was first prepared and sterilized by autoclaving
(Webco GmbH & Co. KG Bad Schwartau,
Phytochemical screening of plant extracts was carried out qualitatively for the presence of terpenoids, steroids, tannins, flavonoids, amino acids, glycosides, saponins, and alkaloids (Harborne, 1998).
Result and Discussion
����������� The dichloromethane, methanolic and aqueous extracts of the whole plant of A. paniculata were investigated at 3 different concentrations by disc diffusion method against 12 bacterial strains notable for causing skin infections. The antibacterial activity was expressed as the average diameter of the zone of inhibition of bacterial growth around the disc. The minimum inhibitory concentration (MIC) of active extracts was determined by using the agar dilution assay. Most of the extracts displayed relatively high antibacterial activity against most of the tested microorganisms with the diameter of inhibition zones ranging between 6.00 � 0.00 to 24.00 � 0.00 (Table1). The gram-positive strains were found to be the most susceptible to growth inhibition by the plant extracts forming zones of inhibition ranging from 7.00 � 0.00 to 24.00 � 0.00 (Table1). The DCM extract was the least potent against S. saprophyticus (7.00 � 0.00) at 250 �g/disc and the methanolic extract revealed the most potent antibacterial activity against E. faecalis (24.00 � 0.00) at 1000 �g/disc (Table1). However, no activity was observed with the DCM, methanolic and aqueous extracts of the plant at 250 �g/disc against M. luteus, S. pyogenes, E. faecalis and S. saprophyticus (Table1). The gram negative strains were less sensitive to the plant extracts as compared to the gram positive, forming zones of inhibition ranging from 6.00 � 0.00 to 20.33 � 0.58 (Table1). The aqueous extract was the least potent against N. meningitis (6.00 � 0.00) at 250 �g/disc and the methanolic extract showed the most potent activity against P. mirabilis (20.33 � 0.58) at 1000 �g/disc (Table1). No activity was observed with the DCM, methanolic and aqueous extracts of the plant at 250 �g /disc against P. aeroginosa and K. pneumoniae (Table1). P. aeroginosa and all Staphylococcus strains used for the study were found to be resistant to tetracycline (Table1).
�������������� The highest MIC value was 300 �g /ml exerted by the DCM extract against M. lutues and B. anthracis and the aqueous extract against M. luteus and E. faecalis respectively. The least MIC was 150 �g/ml exerted by the aqueous extract against S. aureus and the methanolic extract against B. anthracis respectively (Table2). The highest MIC value was found to be 300 �g/ml exerted by the aqueous and DCM extracts against K. pneumonia and P.aeroginosa respectively and the least was 150 �g/ml exerted by the aqueous extract against P. vulgaris and the methanolic extract against N. meningitis respectively (Table 2).
���������������� Flavonoids, alkaloids and glycosides were present in all extracts of Andrographis paniculata. Tannins, amino acids and saponins were present in methanol and aqueous extracts but were absent in dichloromethane extract. However, terpenoids and steroids were found to be present in dichloromethane and methanol extracts and were absent in aqueous extracts (Table 3).
Table 1. In vitro antibacterial activity of dichloromethane, methanol and aqueous extracts of whole plant of Andrographis paniculata. (values are mean of three replicates).
Presence of phytochemicals flavonoids (Roa et al., 2004) and diterpenoid lactones (Reddy et al., 2003) in Andrographis paniculata have been reported.
Antibacterial activity of phytochemical andrographolide, a labdane diterpenoid (Xu et al., 2006) isolated from plant materials has been studied. The antibacterial activity of the polar and non-polar extracts of the whole plant Andrographis paniculata suggests that every extract contains the effective active phytochemicals responsible for the elimination of microorganisms responsible for skin diseases.
Table 2. Minimum inhibitory concentration of of dichloromethane, methanol and aqueous extracts of whole plant of Andrographis paniculata against microorganisms. (values are mean of three replicates).
Table 3. Qualitative analysis of the phytochemicals in the dichloromethane, methanolic and aqueous extracts of Andrographis paniculata.
- = Negative (absent),�� +�� = Positive (present)
����������� In the present era, plant and herb resources are abundant, but these resources are dwindling fast due to the onward march of civilization (Vogel, 1991). Although a significant number of studies have been used to obtain purified phytochemicals, very few screening programmes have been initiated on crude plant materials. It has also been widely observed and accepted that the medicinal value of plants lies in the bioactive phytocomponents present in the plants (Veeramuthu et al., 2008). The greater susceptibility of gram-positive bacteria to plant extracts has been previously reported in South American (Paz et al., 1995), African (Kudi et al., 1999; Vlietinck et al., 1995) and Australian (Palombo & Semple, 2001) medicinal plant extracts. Susceptibility differences between gram-positive and gram-negative bacteria may be due to cell wall structural differences between these classes of bacteria. The gram-negative bacterial cell wall outer membrane appears to act as a barrier to many substances including antibiotics (Tortora et al., 2001). The significant results obtained in our study confirm the antibacterial potential of the plant investigated, and its usefulness in the treatment of skin infections. This in vitro study corroborates the antibacterial activity of A. paniculata used in folkloric medicine to treat skin infections (Jain, 1991; Ahmed et al., 1998). All these extracts were shown to exhibit inhibitory activity against most of the pathogenic bacteria which cause chronic bacterial skin infections. However, they were ineffective at low concentrations against S. saprophyticus, E. faecalis, B. anthracis, M. luteus, S. pyogenes, K. pneumoniae and P. aeroginosa. Hence, their medicinal uses in infections associated with these bacterial species are not recommended. A. paniculata could be a potential source of new antibacterial agents in the treatment of skin infirmities which are associated with these bacteria.
The present study explicitly exhibited the antibacterial effect of various extracts of Andrographis paniculata against skin infections causing bacterial strains. The inhibitory effect of the extracts justified the medicinal use of Andrographis paniculata in the treatment of skin infirmities by traditional practitioners and further study is mandatory to find out the active principles of medicinal value.
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