S. Irshad et al / Research Journal of Biology (2012), Vol. 02, Issue 01, pp.

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Research Paper

In-Vitro Anti-Bacterial Activities of Three Medicinal Plants Using Agar Well Diffusion Method
Saba Irshad1, Maryum Mahmood1 and Farzana Perveen2
2

Institute of Biochemistry and Biotechnology, University of the Punjab, Lahore-54590, Pakistan Chairperson, Department of Zoology, Hazara University, Garden Campus, Mansehra-21300, Pakistan Tel.: +92-42-99230355 ; Fax:+92-42-9923242 E-Mail(s):saba.ibb@pu.edu.pk ; sabairshad2003@yahoo.com

Abstract
Medicinal plants play a vital role for health care. The constituents of medicinal plants control the hyperglycemia as well as its secondary complications. The purpose of this study was to evaluate the antibacterial activity of medicinal plants by agar well diffusion assay. Three medicinal plants (Saussurea lappa, Ricinus communis and Acacia nilotica) were used against four bacterial strains (Lactobacillus subtilis, Bacillus thureogenesis, Corney bacterium and Escherichia coli). These three medicinal plants were macerated into three forms (Ethanol, Chloroform and Simple macerated extracts). Then, with the help of agar diffusion assay the antibacterial activity of these medicinal plants were measured. The zones of inhibition were measured with the help of scale and they were represented by graphs and tables. These medicinal plants showed antibacterial activity against four available bacterial strains. The present study suggests that the ethanol extracts of these medicinal plants contain compounds that can form the basis for the development of a novel broad spectrum antibacterial formulation. Keywords: Saussurea lappa, Acacia nilotica, Ricinus communis, Antibacterial Activity, Agar Diffusion Assay

1. Introduction
The use of medicinal plants is increasing day by day to cure the non-communicable diseases (Latheef et al, 2008). The various chronic diseases such as coronary heart diseases, atherosclerosis, cancer and aging was caused by oxidative damage which occurred due to reactive oxygen species such as super oxide radicals, hydroxyl radicals and peroxyl radicals to lipids, proteins and nucleic acids (Finkel et al, 2000). The intake of fruits and vegetables can cure such type of diseases (Eberhardt et al, 2000). China used medicinal plants for the treatment of many human diseases. Now days people use Chinese medicinal herbs (CMHs) because of their low toxicity and good therapeutic performance. CMHs have also been used as flavours, pigments and foods (Liu et al, 2000). Several studies have shown that these herbal medicines posses more potent antioxidant activities than common dietary plants (Cai, 2004 & Dragland et al, 2004). Saussurea lappa is an endangered medicinal herb. It is found in Himalayas and Kashmir Valley in India. It belongs to the plant family Compositae and genus Saussurea. It is a traditional medicinal plant which is used in many herbal remedies like aphrodisiac, as a stimulant, as a tonic and as an antiseptic. This plant has also anthelmintic, antiseptic, antispasmodic, aromatic, astringent, carminative, diuretic, expectorant, insecticidal, prophylactic, stimulant, tonic action. The roots of Saussurea lappa are also used in herbal preparations. The acylated flavone glycosides are obtained from the roots of the Saussurea lappa and these glycosides are helpful to

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proof the antifungal activity of saussurea lappa (Rao et al, 2007). Acacia nilotica related to family Fabaceae-Mimosoideae. The division of Acacia nilotica is Magnolophyta and class is Magnolipsida. The genus is Acacia and species is nilotica. The ailments treated by this plant include colds, congestion, fever, gallbladder, hemorrhage, hemorrhoids, leucorrhoea, ophthalmic, sclerosis and small pox. Acacia bark is drunk for intestinal pains. Other preparations are used for gargle, toothache, ophthalmic and syphilitic ulcers. The roots of Acacia are used to treat Tuberculosis (Rani et al, 2004). Ricinus communis is related to Euphorbiaceae family. The extract from the leaves in water are used against rheumatism, headache, drops (edeme), abscesses, ringworms and warts. Castor bean is cultivated for the seeds, which yield fast drying; non yellowing oil used mainly in industries and medicines. The oil of Ricinus communis is also used in coating fabrics and other protective coverings in the manufacture of high grade lubricants, transparent, typewriter and printing inks in textile dyeing (Vermeer et al, 2003). The stem of castor beans is used into paper and wallboard (Hill et al, 1996).

2.4. Maceration Maceration is also called cold extraction. The process of maceration was used to separate the different constituents and ingredients of the plants in the form of crude extract. The three medicinal plants were macerated by three different ways, in which different solvents were used. Maceration with Ethanol; Maceration with Chloroform and Maceration with distilled water (Simple maceration). In the process of maceration with solvents either Ethanol or Chloroform, each weighed crushed medicinal plants were dissolved in 150ml of solvent. The crude extract was placed at magnetic stirrer for equal mixing. After that, these crude extracts were allowed to remain at room temperature for four days. Then, each extract was filtrated under fume hood. The filtered extract was allowed to dry at room temperature for 10 minutes. Furthermore, each extract was preserved for further procedure. 2.5. Agar Diffusion Assay 100 ml conical flask of nutrient broth was inoculated with the test organisms and incubated at 37C for overnight. By using a sterile pipette, 0.6ml of the broth culture of each test organism was added to 60ml of molten agar, which were cooled at 45C. Mixed well and poured into a sterile Petri plate. 0.2ml of culture was added to 20ml of agar. Agar test plates of each test organism were prepared. The agar was allowed to set and harden and required numbers of holes were cut using a sterile cork borer ensuring proper distribution of holes in the periphery and one in the centre. Agar plugs were removed. Different cork borers were used for different test organisms. Canamycin was used in comparison with antibacterial activity of other test organisms. 1mg of canamycin was dissolved in 1ml of triple distilled water. In each hole, sample was loaded. In central hole, 200l of bacterial strain was loaded. In one hole, control was loaded and in the remaining holes 100l of control and 100l of drug was loaded. Then, the plates were left at room temperature for 2 hours to allow diffusion of test sample and incubated face upwards at 37C for overnight. The diameter of the zones of inhibition was measured with scale.

2. Materials and Methods

The present research work was designed to evaluate antibacterial activity of medicinal plants including Saussurea lappa, Acacia nilotica and Ricinus communis. This study was performed in The Institute of Biochemistry and Biotechnology, University of the Punjab, Lahore. In this study, there were four steps which are as follows: 2.1. Collection of Medicinal Plants There were three medicinal plants which were collected from different areas. The names of these test samples are Saussurea lappa (Kuth), Acacia nilotica (Common seaweed) and Ricinus communis (Castor bean). The bacterial strains used in this project were Lactobacillus subtilis, Bacillus thureogenesis, Corney bacterium and Escherichia coli. 2.2. Identification of Medicinal Plants The medicinal plants were identified by Dr. Nassir (Assistant Professor) from the department of Botany. 2.3. Crushing of Medicinal Plants 50g of each plant was weighed and then crushed in sterile pestle mortal.

3. Results
It was detected that Saussurea lappa (Kuth) and Acacia nilotica (Common seaweed) had ability to show antibacterial activity against Bacillus thureogenesis, Corney bacterium and Escherichia coli without the combination of drugs. Each well contained 400l of macerated plant. The three macerated forms of plants (Ethanol, Chloroform and Simple macerated) were used. Different concentrations of macerated plants (0.9mg, 1mg, 5mg, 10mg and 20mg) were used in 1ml of Ethanol and

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Chloroform. Some wells contained bacterial strains. Thus, the holes which contained concentration of medicinal plant inhibited the growth of bacteria while other wells showed contamination. The concentration which was below 1mg also showed contamination. The zones of inhibition (cm) were measured in table1-8. The concentration (mg/ml) was measured along x-axis while zones of inhibition (cm) were measured along y-axis (Figure1-8). These figures showed antibacterial activity of Saussurea lappa, Acacia nilotica against Bacillus thureongenesis, Escherichia coli, Corney

bacterium and Lactobacillus subtilis. Ricinus communis (Castor bean) showed antibacterial activity against Bacillus thureogenesis, Corney bacterium and E.coli without the combination of drugs. The zones of inhibition (cm) were measured (Table 9-11). The concentration (mg/ml) was measured. It was observed that Ricinus communis showed antibacterial activity against the above three bacterial strains (Figure 9-11).

Table 1. Zones of Inhibition of Different Concentrations of Macerated Saussurea lappa with Bacillus thureogenesis

Ethanol Macerated Plant Concentration (mg/ml) Zone of Inhibition (cm) 1 0.3 5 0.5 10 0.7 20 1 1 0.3

Chloroform Macerated Plant 5 0.4 10 0.6 20 0.9 1 0.2

Simple Macerated Plant 5 0.3 10 0.5 20 0.7

1.2 Z o n e o f In h i b i t io n ( c m ) 1 0.8 0.6 0.4 0.2 0 1 5 10 20 1 5 10 20 1 5 10 20 Z o n e o f In h i b i t io n ( c m )

1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 1 5 10 1 5 10 1 5 10

Concentration (mg/ml)
Figure 1. Ethanol, Chloroform and Simple Macerated Saussurea lappa with Bacillus thureogenesis

Concentration (mg/ml)
Figure 2. Ethanol, Chloroform and Simple Macerated Saussurea lappa with E.coli

Table 2. Zones of Inhibition of Different Concentrations of Macerated Forms of Saussurea lappa with E.coli

Ethanol Macerated Plant Concentration (mg/ml) Zone of Inhibition (cm) 1 0.4 5 0.6 10 0.9 1

Chloroform Macerated Plant 5 0.6 10 0.8

Simple Macerated Plant 1 0.3 5 0.5 10 0.7

0.3

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Table 3. Zones of Inhibition of Different Concentrations of Macerated Plants of Kuth with Corny bacterium

Ethanol Macerated Plant Concentration (mg/ml) Zone of Inhibition (cm) 1 0.4 5 0.5 10 0.9 1

Chloroform Macerated Plant 5 0.5 10 0.6 1

Simple Macerated Plant 5 0.4 10 0.5

0.4

0.3

Table 4. Zones of Inhibition of Different Concentrations of Macerated forms of Saussurea lappa with Lactobacillus subtilis

Ethanol Macerated Plant Concentration (mg/ml) Zone of Inhibition (cm) 1 0.5 5 0.8 10 1

Chloroform Macerated Plant 1 0.4 5 0.5 10 0.7 1

Simple Macerated Plant 5 0.4 10 0.6

0.3

Table 5. Zones of Inhibition of Different Concentrations of Macerated Acacia nilotica with Bacillus thureogenesis

Ethanol Macerated Plant Concentration (mg/ml) Zone of Inhibition (cm) 1 0.3 5 0.6 10 0.8 20 1 1 0.3

Chloroform Macerated Plant 5 0.5 10 0.7 20 0.8 1 0.2

Simple Macerated Plant 5 0.4 10 0.6 20 0.7

1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 1 5 10 1 5 10 1 5 10

1.2 Z o n e o f In h ib it io n ( c m ) 1 0.8 0.6 0.4 0.2 0 1 5 10 1 5 10 1 5 10

Z o n e o f In h i b i t io n ( c m )

Concentration (mg/ml)
Figure 3. Ethanol, Chloroform and Simple Macerated Sussurea lappa with Corny bacterium

Concentration (mg/ml)
Figure 4. Ethanol, Chloroform and Simple Macerated forms of Saussurea lappa with Lactobacillus subtilis

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Table 6. Zones of Inhibition of Different Concentrations of Macerated Acacia nilotica with Corny bacterium

Ethanol Macerated Plant Concentration (mg/ml) Zone of Inhibition (cm) 1 0.4 5 0.6 10 0.9 1 0.4

Chloroform Macerated Plant 5 0.5 10 0.8 1

Simple Macerated Plant 5 0.5 10 0.7

0.3

Table 7. Zones of Inhibition of Different Concentrations of Macerated Acacia nilotica with E.coli

Ethanol Macerated Plant Concentration (mg/ml) Zone of Inhibition (cm) 1 0.3 5 0.5 10 0.8 1 0.3

Chloroform Macerated Plant 5 0.5 10 0.7 1

Simple Macerated Plant 5 0.4 10 0.7

0.2

Table 8. Zones of Inhibition of Different Concentrations of Macerated Forms of Acacia nilotica with Lactobacillus subtills

Ethanol Macerated Plant Concentration (mg/ml) Zone of Inhibition (cm) 1 0.4 5 0.6 10 1 1 0.4

Chloroform Macerated Plant 5 0.5 10 0.9 1

Simple Macerated Plant 5 0.4 10 0.7

0.3

1.2 Z o n e o f In h i b i t io n ( c m ) 1 Z o n e o f In h ib it io n ( c m )

0.8 0.6 0.4 0.2 0 1 5 10 20 1 5 10 20 1 5 10 20

1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 1 5 10 1 5 10 1 5 10

Concentration (mg/ml)
Figure 5. Ethanol, Chloroform and Simple Macerated Acacia nilotica with Bacillus thureogenesis

Concentration (mg/ml)
Figure 6. Ethanol, Chloroform and Simple Macerated Acacia nilotica with Corny bacterium

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Table 8. Zones of Inhibition of Different Concentrations of Macerated Forms of Acacia nilotica with Lactobacillus subtills

Ethanol Macerated Plant Concentration (mg/ml) Zone of Inhibition (cm) 1 0.4 5 0.6 10 1 1 0.4

Chloroform Macerated Plant 5 0.5 10 0.9 1

Simple Macerated Plant 5 0.4 10 0.7

0.3

Table 9. Zones of Inhibition of Different Concentrations of Macerated Forms of Ricinus communis with Bacillus thureogenesis

Ethanol Macerated Plant Concentration (mg/ml) Zone of Inhibition (cm) 1 0.3 5 0.6 10 0.8 20 1 1 0.3

Chloroform Macerated Plant 5 0.5 10 0.7 20 0.8 1 0.2

Simple Macerated Plant 5 0.4 10 0.6 20 0.7

Table 10. Zones of Inhibition of Different Concentrations of Macerated Ricinus communis with Corny bacterium

Ethanol Macerated Plant Concentration (mg/ml) Zone of Inhibition (cm) 1 0.5 5 0.6 10 0.8 1

Chloroform Macerated Plant 5 0.5 10 0.7

Simple Macerated Plant 1 0.2 5 0.5 10 0.7

0.4

0.9 Z o n e o f In h ib it io n ( c m ) Z o n e o f In h ib it io n ( c m ) 1 5 10 1 5 10 1 5 10 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0

1.2 1 0.8 0.6 0.4 0.2 0 1 5 10 1 5 10 1 5 10

Concentration (mg/ml)
Figure 7. Ethanol, Chloroform and Simple Macerated Acacia nilotica with E.coli

Concentration (mg/ml)
Figure 8. Ethanol, Chloroform and Simple Macerated Acacia nilotica with Lactobacillus subtills

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Table 11. Zones of Inhibition of Different Concentrations of Macerated Ricinus communis with E.coli

Ethanol Macerated Plant Concentration (mg/ml) Zone of Inhibition (cm) 1 0.4 5 0.6 10 1 1

Chloroform Macerated Plant 5 0.5 10 0.8

Simple Macerated Plant 1 0.2 5 0.5 10 0.7

0.3

1.2 1 Z o n e o f In h ib it io n ( c m ) 1 5 10 20 1 5 10 20 1 5 10 20 Z o n e o f In h i b i t io n ( c m )

0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 1 5 10 1 5 10 1 5 10

0.8 0.6 0.4 0.2 0

Concentration (mg/ml)
Figure 9. Ethanol, Chloroform and Simple Macerated Forms of Ricinus Communis with Bacillus thureogenesis

Concentration (mg/ml)
Figure 10. Ethanol, Chloroform and Simple Macerated Forms of Ricinus communis with Corny bacterium

1.2 Z o n e o f In h ib it io n ( c m ) 1 0.8 0.6 0.4 0.2 0 1 5 10 1 5 10 1 5 10

(Joy et al, 1998). Agar diffusion assay were used for evaluation of antibacterial activity of these three medicinal plants. This method had already been used for antibacterial activity of Neem, Peppermint and Aloe Vera (Irshad et al, 2011a & b) Saussurea lappa having antibacterial activity against four bacterial strains (Lactobacillus subtilis, Bacillus thureogenesis, Corney bacterium and Escherichia coli). This study is correlated with the work of Yang in which he studied the antibacterial activity of Saussurea lappa with Escherichia coli and Bacillus strains (Yang et al, 2004). It is clear from the present study that Ethanol is most effective against bacterial strains than the other macerated forms. The antibacterial activity of Acacia nilotica evaluated against four bacterial strains (Lactobacillus subtilis, Bacillus thureogenesis, Corney bacterium and Escherichia coli). Relativity of effectiveness of crude extract were same as above plants i.e. Ethanol extract is most effective one while Chloroform extract is more effective than simple macerated form (Rani et al, 2004). Seeds of Acacia nilotica were used to check its antibacterial activity. Acacia nilotica is very effective against diarrhoeal diseases (Agunu et al, 2005).

Concentration (mg/ml)
Figure 11. Ethanol, Chloroform and Simple Macerated Ricinus communis with E.coli

4. Discussion
Plants used in Ayurveda can provide biologically active molecules and lead structures for the development of modified derivatives with enhanced activity and /or reduced toxicity of the 2,50,000 higher plant species on earth, around 5000 species have specific therapeutic value

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Ricinus communis showed antibacterial activity against Bacillus thureogenesis, Corney bacterium and Escherichia coli. The Ethanol macerated extract was most effective against bacterial strains. The seeds of Castor bean were used to check the antibacterial activity in this study. In the seeds, a compound ricin is present which is toxic and inhibit the growth of bacteria (Brzezinski et al, 2007). Another compound ricinoleate is present in seeds which are toxic and Castor oil has 90% of its composition (McKeon et al, 2000).

Finkel, T., and Holbrook, N. (2000) Evaluation of two methods for the extraction of antioxidants from medicinal plants. Journal of Nature, 408, pp. 239-247. Hill, A., Cane, D., Mau, C., and West, C. (1996) High level expression of Ricinus communis casbene synthase in E.Coli and characterization of recombinant enzyme. Journal of Arch. Biochem. Biophys., 336, pp. 283-285. Irshad, S., Butt, M., and Younus, H. (2011 a) In-Vitro Antibacterial activity of Two Medicinal Plants Neem (Azadirachta indica) and Peppermint. International Research Journal of Pharmaceuticals, 01(01), pp. 1-6. Irshad, S., Butt, M., and Younus, H. (2011 b) In-Vitro Antibacterial activity of Aloe Barbadensis Miller (Aloe Vera). International Research Journal of Pharmaceuticals, 01(02), pp. 60-65. Joy, P.P., Thomas, J., Mathew, S., and Skari, B.P. (1998) Medicinal Plants. Kerala Agriculture University, Aromatic and Medicinal Plants Research Station, 33, pp. 33-133. Latheef, S.A., Prasad, B., Bavaji, M., and Subramanyam, G. A. (2008) Database on endemic plants at Tirumala hills in india. Journal of Bioinformation, 2, pp. 260-262. Liu, F., and Ng, T. B. (2000) Evaluation of two methods for the extraction of antioxidants from medicinal plants. Journal of Life Sci., 66, pp. 725-735. McKeon, T., Chen, G., and Lin, J. (2000) Biochemical aspects of castor oil biosynthesis. Journal of Biochem. Soc. Trans., 28, pp. 972-974. Rani, P., and Khullar, N. (2004) Antimicrobial evaluation of some medicinal plants for their anti-enteric potential against multi drug resistance Salmonella typhi. Journal of Inter Science, 18, pp. 670-673. Rao, K., Babu, G., and Ramnareddy, Y. (2003) Acylated flavone glycosides from the roots of Saussurea lappa and their antifungal activity. Journal of Molecules, 12, pp. 328-344. Vermeer, C. P., Nastold, P., and Jetter, R. (2003) Homologous very-long-chain 1,3-alkanediols and 3hydroxyaldehydes in leaf cuticular waxes of Ricinus communis L. Journal of Phytochemistry, 62, pp. 433438. Yang, M., Wang, C. M., Zhang, Q., Han, Y. F., and Jia, Z. (2004) Sesquiterpene, lignans and other constituents from Saussurea macrota. Journal of Pharmazie., 59, pp. 972976.

5. Conclusion
The results of present study support the traditional usage of the studied plants and suggest that some of the plant extracts possess compounds with antimicrobial properties that can be used as antimicrobial agents in new drugs for the therapy of infectious diseases caused by pathogens. The most active extracts can be subjected to isolation of the therapeutic antimicrobials to carry out further pharmacological evaluation.

Acknowledgements
Authors wish to thank Muhammad Shahbaz, Islamia University, Bahawalpur, Pakistan and Muneeba Butt, Institute of Biochemistry and Biotechnology, University of the Punjab, Lahore for their technical assistance.

References
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