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Proceedings of the National Seminar on Current Concepts in immunology, 18th and 19thAugust 2011

Antibacterial Activity of Medicinal Plants against Human Pathogenic Bacteria

Linu Susan Jacob and Shirly Annie Oommen Post graduate & Research Department of Zoology, St.Thomas College Kozhencherry, Kerala, India. ABSTRACT The Drugs of natural origin play an important role in health care system from ancient time onwards. Most people rely on antibiotics for their health needs. Their excess uses result in Multiple Drug Resistance (MDR) and Adverse Drug Resistance (ADR) in microorganisms. So it is very important to select medicinal plants with antibacterial properties for human consumption against different pathogens. The present study was to investigate the antibacterial activities of five medicinal plants, Aloe vera, Arrow root, Gamboge, Neem and Nutmeg at four different concentrations (25%, 50%, 75% and 100%) against five human pathogens namely Escherichia coli, Salmonella typhi, Salmonella paratyphi, Vibrio cholerae and Vibrio parahaemolyticus. Their antibacterial activities were compared with that of five widely used antibiotics (Ampicillin, Amoxicillin, Ciprofloxacin, Kanamycin and Streptomycin). Among the five medicinal plants tested 100% and 75% extract of all medicinal plants showed resistance against five bacterial species. Antibacterial activities showed by medicinal plants were species specific and showed more action against the tested antibiotics. Aloe vera, Arrow root and Neem showed maximum inhibition zone to Salmonella typhi, while the treatment with Gamboge extract on Salmonella paratyphi showed highest inhibition. Maximum activity of Nutmeg extract for Escherichia coli. Different pathogens responded differently to plant extracts at different concentrations. The antibacterial study on plant extracts demonstrated that folk medicine can be as effective as modern medicine to combat human pathogens and can be used as an alternative to treat infectious diseases. Key words: Aloe Vera, Arrow root, Gamboge, Neem, Nutmeg, Bacterial strains, zone of inhibition, Antibiotics, antibacterial activity. INTRODUCTION Since prehistoric times medicinal plants are used as natural medicines. The drugs of natural origin play an important role in health care particularly in rural areas of India. India is the home for a number of commercially important medicinal plants and aromatic crops. Studies involving traditional medicines frequently contribute new ideas. (Samy and Gopalakrishnakone, 2010) There is a need to introduce new sources and if possible from less employed regions and habitats to maximize the discovery of novel bioactive metabolites. Multiple Drug Resistance (MDR) has developed due to excess use of antimicrobials and re-emergence of diseases. Adverse Drug Resistance (ADR) and high cost of antimicrobials have been played important part in ineffective management of infectious diseases in many developing countries. Natural products of higher plants may give a new source of antimicrobial agent with novel mechanisms of action (Chandrappa et al., 2010). Many indigenous systems such as Ayurveda, Yoga, Unani, Homeopathy, Naturopathy and Siddha are famous and prevailing in India from decades (Vaidya and Devasagayam, 2007). Clinical and therapeutic uses of many Indian medicinal plants have been recorded (Claeson et al., 2000). Herbal drug industries, it is a very fast growing sector in the international market but India has not up to the mark due to lack of scientific input and approaches in the herbal world (Bhutani and Gohil, 2010). Nicholas Piramal Industries Ltd (NPIL), one of the major pharma players in India has built up a plant extract library having 6000 extracts prepared from around 2300 plant species collected from all over India and such libraries could serve as a powerful tool and source of extracts to be screened for biological activities using high-throughput assays (Jachak and Saklani, 2007). It has also have been reported that by using Reverse pharmacological approach several institutes are carrying clinical research on the potential health benefits of herbal drugs (Vaidya and Devasagayam, 2007; Patwardhan et al., 2004). MATERIALS AND METHODS Collection of samples: The medicinal plants used for the experiment were of Aloe vera (Aloe vera), Maranta arundinacea (Arrow root) , Garcinia gummi-gutta (Gamboge), Azadirachta indica (Neem) and Myristica fragrans (Nutmeg).They were chosen depending on their wide use and popularity in the region.

Collection of test organisms and preparation of stock culture: Test organisms were received from School of Environmental Sciences, M.G. University, Kottayam and School of Medical Education, Arpookkara and sub cultured in appropriate selective media. Collection of antibiotics: Antibiotics such as Ampicillin (25mcg), Amoxicillin (30mcg), Ciprofloxacin (30mcg), Kanamycin (30mcg) and Streptomycin (25mcg) were purchased (Hi-Media). Preparation of medicinal plant extracts: The medicinal plants were obtained fresh from the surroundings. They were cleaned, descaled wherever necessary, washed in sterile distilled water. In order to obtain the medicinal plant extracts, about 100g of washed plants were crushed with mortar and pestle. The extracts were later sieved through a fine mesh cloth. These extracts were considered as the 100% concentration and from these different concentrations such as 75%, 50% and 25% were made by diluting the crude extracts with appropriate volumes of sterile distilled water. Preparation of Inoculum: The selected strains of bacteria were ascetically inoculated into 10ml of sterile nutrient broth. The nutrient broth tubes were then incubated at 37C for 16-18 hours. All the bacterial strains showed very good as indicated by the turbidity. Antibacterial sensitivity testing using filter paper method: Filter paper disc of 8 mm were prepared and sterilized. Using ethanol dipped and flamed forceps, these discs were aseptically placed over nutrient agar plates seeded with the respective test microorganisms (Srinivasan et al., 2001).Control discs are prepared by incorporating discs with 100l distilled water. Hundred l of various medicinal plant extract (100%) were prepared and from these different concentrations such as 75%, 50% and 25% were made. They were aseptically incorporated into these discs. The plates were incubated in upright position at 37C for 24 hrs. After incubation diameter of inhibition zone were measured in mm and results were recorded. Diameter of inhibition zones less than 12 mm were not considered as having any antibacterial activity. Antibiotic sensitivity testing: The test microorganisms were tested for their sensitivity against various antibiotics such as Ampicillin (25mcg), Amoxicillin (30mcg), Ciprofloxacin (30mcg), Kanamycin (30mcg) and Streptomycin (25mcg) by their disc-diffusion method (Bauer et al., 1966).The cultures were enriched in sterile nutrient agar broth for 6-8 hrs. Using sterile cotton swabs, the cultures were aseptically swabbed over sterile nutrient agar plates. Using ethanol dipped flamed forceps, the antibiotic discs were aseptically placed over the seeded nutrient agar plates sufficiently separated from each other so as to avoid overlapping of the inhibition zones. The plates were incubated at 37C for 24 hrs and the diameter of the inhibition zones were measured in mm. RESULTS The inhibitory activities of antibiotic such as Ampicillin, Amoxicillin, Ciprofloxacin, Kanamycin and Streptomycin against five bacterial pathogens E.coli, Salmonella typhi, Salmonella paratyphi, Vibrio cholerae and Vibrio parahaemolyticus are given in Table 1 and figure 1. Table 2 and Fig. 2 gives the zone of inhibition of A. vera for 100% extract is more than that of Ampicillin and Amoxicillin, but it is less effective than the other three antibiotics used, such as Ciprofloxacin, Kanamycin and Streptomycin. Arrow root extracts showed high antibacterial activity against S. typhi at 100% and 75% and V. cholerae at 100%. 50% concentration of arrow root extract showed moderate antibacterial activity against S. typhi. Different bacterial species responded differently to the arrow root extract. The arrow root extract showed more effect than antibiotics, Ampicillin, Amoxicillin and Kanamycin used for controlling the growth of infection caused by S. typhi and V. cholerae, whereas, E. coli and V. parahaemolyticus has no effect with the arrow root extract (Table 3 and fig. 3). The inhibition zone obtained by all concentrations of Gamboge (25%, 50%, 75% and 100%) extracts when compared with the control showed high bacterial resistance (Table 4, fig. 4). The antibacterial activity of neem extract against the five bacteria represented in Table 5 and fig. 5 showed that 50%, 75% and 100% neem extracts showed high resistant activity against E. coli, when compared with the sterile disc, whereas, it is less active than Ciprofloxacin and Streptomycin. In the case of S. typhi, S. paratyphi, V. cholerae and V. parahaemolyticus the neem extract exhibit more resistant against Ampicillin and Amoxicillin. Both 75% and 100% neem extract showed antibacterial activity for the selected bacterial species and the activity was varying with respect to the concentration. Among the five selected bacterial species, E. coli and V. cholerae are highly sensitive to the nutmeg extract (100%). The 75% and 50% nutmeg extract also showed antibacterial activity against five selected bacterial species (Table 6, fig.6). 100% extract was highly effective when compared with antibiotics such as Ampicillin, Amoxicillin and Kanamycin. Antibacterial activity for 100% concentration represented in Table 7& Fig.7 divulged that the Aloe vera extract showed maximum zone of inhibition to S.typhi (28mm) followed by V. parahaemolyticus (24mm) and minimum to V. cholerae. (11mm).The extracts of Arrow root showed maximum zone of inhibition for S.typhi and least to E.coli (9mm), whereas the other three bacteria having moderately high degree of antibacterial activity. While the treatment with Gamboge extract, S.paratyphi showed the highest zone of inhibition (29mm) and minimum for E.coli (20mm), but all the plant extracts exhibit antibacterial activity for the 100% Gamboge extracts. The highest zone of inhibition was noticed in S.typhi with the neem extract (28mm) and lowest for S.paratyphi (21mm).The maximum activity was observed to nutmeg extract for E.coli and minimum zone of inhibition was V.parahaemolyticus (11mm).

Table1. Antibacterial activity of antibiotics against various bacteria: Figure 1. Average diameter of inhibition zone(mm) by Antibiotics (n=3) Amp Amo Ciprofl Kanam Strept icilli xicil oxacin ycin omyci n lin n 9 11 42 23 37 12 10 9 8 13 12 7 8 37 41 40 46 28 28 29 24 46 25 36 37
Antibiotis

Zone of inhibition zone (mm)

Bacteria

E. coli S.typhi S. paratyphi V.cholerae V.parahaemolyticus

50 45 40 35 30 25 20 15 10 5 0

Bacteria E. coli S.typhi S. paratyphi V.cholerae V.parahaemolyticus

Table2. Antibacterial activity of Aloe Vera against various bacteria:

Figure 2.

Bacteria

Control

Zone of inhibition zone(mm)

E. coli S.typhi S. paratyphi V.cholerae V.parahaemolyticus

Average Diameter of inhibition zone(mm) at different concentrations ( n = 3 ) 100% 75% 50% 25% 19 7 6 6 28 12 11 24 12 10 10 13 10 9 9 10 7 6 7 9

6 10 9 16 10

30 25 20 15 10 5 0

100% 75% 50% 25% Control

Bacteria

Table3. Antibacterial activity of Arrow root against various bacteria: Bacteria Average Diameter of inhibition zone(mm) at different concentrations (n = 3) 100% 75% 50% 25% 9 8 7 6 29 13 20 9 20 11 15 8 12 9 12 7 9 8 8 6 Control

Figure 3

E. coli S.typhi S. paratyphi V.cholerae V.parahaemolyticus

6 10 9 16 10 Figure 4

Table4. Antibacterial activity of Gamboge against various bacteria:

Bacteria

E. coli S.typhi S. paratyphi V.cholerae V.parahaemolyticus

Average Diameter of inhibition zone(mm) at different concentrations (n = 3) 100% 75% 50% 25% 20 18 15 13 24 19 16 12 29 16 12 10 23 15 13 11 21 19 18 12

Control

6 10 9 16 10

Table5. Antibacterial activity of Neem against various bacteria: Bacteria Average Diameter of inhibition zone(mm) at different concentrations (n = 3) 100% 75% 50% 25% 22 17 10 7 28 22 15 10 21 18 14 10 22 16 15 6 24 13 10 9 Control

Figure 5.

E. coli S.typhi S. paratyphi V.cholerae V.parahaemolyticus

6 10 9 16 10

Table6. Antibacterial activity of Nutmeg against various bacteria: Bacteria Average Diameter of inhibition zone(mm) at different concentrations (n = 3) 100% 75% 50% 25% 27 15 9 8 13 10 9 8 13 11 10 8 26 25 24 12 11 10 9 8 Control
Zone of inhibition (mm)

Figure 6.

E. coli S.typhi S. paratyphi V.cholerae V.parahaemolyticus

6 10 9 16 10

30 25 20 15 10 5 0

100% 75% 50% 25% Control

Bacteria

Table 7.Antibacterial activity of five medicinal plant extracts against different human pathogens at 100% concentration: Bacterial pathogen Average Diameter of inhibition zone(mm) against various medicinal plant extracts (n=3) Aloe Arrow Gambo Neem Nutme Vera Root ge g

Figure 7.

Cont rol

E. coli S. typhi S. paratyphi V.cholerae V.parahaemolyticus

19 28 12 11 24

9 29 13 20 25

20 24 29 23 21

22 28 21 22 24

27 13 13 26 11

6 10 9 16 10

DISCUSSION The beneficial medicinal effects of plant materials typically result from the secondary products present in the plant although, it is usually not attributed to a single compound but a combination of the metabolites. The medicinal actions of plants are unique to a particular plant species or group, consistent with the concept that the combination of secondary products in a particular plant is taxonomically distinct (Parekh et al., 2005). Plant essential oils and extracts have been used for many thousands years, in food preservation, pharmaceuticals, alternative medicine and natural therapies. It is necessary to investigate those plants scientifically which have been used in traditional medicine to improve the quality of healthcare. Plant extracts are potential sources of novel antimicrobial compounds especially against bacterial pathogens. In vitro studies in this work showed that the plant extracts inhibited bacterial growth but their effectiveness varied. The antimicrobial activity of many plant extracts had been previously reviewed and classified as strong, medium or weak (Zaika, 1998). An important characteristic of plant extracts and their components is their hydrophobicity, which enable them to partition the lipids of the bacterial cell membrane and mitochondria, disturbing the cell structures and rendering them more permeable. The inhibition produced by the plant extracts against particular organism depends upon the various intrinsic and extrinsic factors (Joshi et al., 2011).

In classifying the antibacterial activity as Gram-positive and Gram negative, it would generally be expected that much greater number would be active against Gram-positive than Bacteria Gram negative bacteria, however in our present study all the bacterial species selected were Gram negative. However, there are some contradictory reports on sensitivity of Gram positive and Gram- negative bacteria. Zaika, 1988 proposed that Gram-positive bacteria are more resistant than Gramnegative bacteria to the antibacterial properties of plant essential oils, while Dorman and Deans (2000) hypothecated that susceptibility of bacteria to plant essential oils and the Gram reaction have little influence on growth inhibition. Lower susceptibility of Gram negative bacteria may be related to the outer membrane of Gram-negative bacteria, which provides the bacterial surface with strong permeability barrier (Nikaido and Vaara, 1985). According to Abu-Shanab et al., (2004) gram negative bacteria are more resistant to plant extracts .This may be due to the permeability barrier provided by the cell walls or to the membrane accumulation mechanism. This is in support of the present findings which showed all the five plant extract Aloe Vera, Arrow root, Gamboge, Neem and Nutmeg was effective on the growth of five human pathogens selected for the study such as E.coli, Salmonella typhi, S.paratyphi, Vibrio cholerae and V. parahaemolyticus. Aloe vera is applied for wound treatment, skin care, tonic drink etc. the active ingredient in A.vera is aloin (an anthrone-C glucoside) as main laxative compound, the gel contains complex polysaccharides, amino acids, minerals, glycoproteins , salicylic acid and enzymes and acetylated mannan. Acemannan is a complex carbohydrate with immune stimulating and antiviral properties. Certain lecitins, which are found in the Aloe pith, are assumed to help in stimulation of immune response by increasing the production of lymphocytes that are known to kill bacteria and some tumor cells. These products, in addition, have uronic acids that are natural detoxicants and take part in the healing process by stripping toxic materials of their harmful effects (Coopoosamy and Magwa, 2007). The antibacterial activity has been attributed to the presence of some active constituents in the extracts. The phytochemical analysis of A.indica had earlier been reported (Kraus, et al., 1981). A.indica in the present study also revealed the presence of terpenes and glycosides. Study suggested a number of active constituents might be present in the neem extract to control the activity of the growth of bacterial species or the might have the antibacterial properties. Salmonella spp. which infects a number of animal species and S.typhi, which causes typhoid has also been tested against plant extract and found to be effective. Intensive use of antibiotics often resulted in the development of resistant strains, these create a problem in treatment of infectious diseases, furthermore antibiotics sometimes associated with side effects(Cunha, 2001), whereas there are some advantages of antimicrobial compounds of medicinal plants such as often fewer side effects, better patient tolerance, relatively less expensive, acceptance due to long history of use and being renewable in nature(Vermani and Garg, 2002).Because of this, the search for new antibiotics continues unabated. These findings support the traditional knowledge of local users and it is a preliminary, scientific, validation for the use of these plants for antibacterial activity to promote proper conservation and sustainable use of such plant resource. 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