In vitro antibacterial activity of Tabernaemontana alternifolia (Roxb) stem bark aqueous extracts against clinical isolates of methicillin resistant Staphylococcus aureus

Infectious diseases are one of the world’s leading causes of premature deaths [1]. Antibiotics which are widely used for the treatment of infectious diseases are under constant threat due to the emergence of antibiotic resistant pathogens such as methicillin resistant Staphylococcus aureus (MRSA), multidrug resistant Pseudomonas aeruginosa, vancomycin resistant Enterococcus (VRE) and multidrug resistant Mycobacterium tuberculosis[2–6]. Among these, MRSA has emerged as agents causing increasing threat of nosocomial infections, more people have died of MRSA infection in US hospitals than of HIV/AIDS and tuberculosis combined during 2007–2008 [7, 8]. Hence, search for novel antimicrobial compounds or alternative therapy for these infections is inevitable.

Plant based medicines are widely used and form an integral part of primary health care in many developing countries across the globe [9–12]. Recently plants have been explored to obtain crude natural extracts for testing and further refinement to develop effective antimicrobial drugs. In India there are different systems of medicine practiced like Ayurveda, Siddha, Unani, Amchi and local health traditions, which utilize a large number of plants or herbs for the treatment of human diseases [13].

Many plant species widely explored for antimicrobial compounds fall into the family Apocynaceae. Within this family, the genus Tabernaemontana is well documented for its biological activities such as antioxidant [14], anticancer [15–17], antifertility/contraceptive [18, 19], anti-pyretic and anti-inflammatory [17, 20], anti-mycobacterial [14], and antimicrobial agent [21–23].

T. alternifolia (Roxb), also known as Ervatamia heyneana (Wall) or T. heyneana (Wall) is endemic to India and found in Goa, Karnataka, Kerala, Maharashtra and Tamil Nadu states [24]. Traditionally a therapeutic preparation made from leaf and stem powder of T. alternifolia (Roxb) in combination with stem bark of Ficus racemosa, Ficus benghalensis, Madhuca longifolia and Strychnos nux-vomica is used to treat skin infections [25]. The other plants, except T. alternifolia, used in this preparation have reported antibacterial activity, but despite the traditional use of T. alternifolia in treatment of skin infections, no scientific report has focused on establishment of the antimicrobial activity of the plant against pathogen [26–29]. We therefore, under took this study to evaluate the antimicrobial potential of the extracts of stem bark of T. alternifolia against pathogens causing skin infections, especially MRSA and VRSA.

Plant derived medicines have been a part of traditional health-care in most parts of the world and the antimicrobial property of plant-derived compounds is well-documented [38]. For the first time here, we established the antimicrobial activity of crude aqueous extract of T. alternifolia against MRSA and VRSA. A study conducted by Duraipandiyan et al. in 2006, showed that the methanol and hexane extract of stem bark of T. alternifolia did not exhibit any antibacterial activity [25]. These observations related to activity may be attributed to the fact that different compounds from the plant material get extracted in solvents of different polarities. Moreover, the fact that the antimicrobial activity of both the SAE and DAE was similar (as indicated by the MIC values) demonstrates that the active principle in the plant stem bark was not extracted in less polar solvents like hexane, ether, methanol, ethyl acetate but got extracted in distilled water. During the extraction process the active principle within the extract survived boiling at 100°C for 6 hours suggesting that it is heat stable. Such a heat stable active principle has been previously reported from other plants [39, 40].

Several studies have focused on establishment of antibacterial activity of the plant extracts but have not focused on showing the time dependent stability of its activity [22, 23, 39]. Here we report that the activity of the extract is stable over a period of 2 months after extraction. This indicates that the active principle present in the aqueous extract of T. alternifolia stem bark has a long shelf life in crude preparation. For a crude extract to be used for topical applications it is mandatory that the extract does not exhibit any cytotoxic activity. Although anticancer alkaloid camptothecin has been isolated from T. alternifolia; aqueous extracts in this study did not show presence of camptothecin (data not shown) [16]. The aqueous extracts did not exhibit any significant cytotoxic effect against Vero cell line (Figure 2); this is consistent with the absence of camptothecin in the extracts. On exposure to SAE, the viable cell counts of Vero cells were observed to be higher as compared to control, suggesting that SAE may have a probable positive effect on cell proliferation. The toxic compound/s present in the stem bark were probably extracted in less polar solvents during sequential extraction, thus, explaining the observed positive effect on cell proliferation only on SAE treatment.

The antimicrobial activity of the crude extract may be attributed to a specific compound or a combination of compounds. The knowledge about the chemical profile of the extract helps in explaining the observed activity and designing experiments for activity fractionation for isolation of the active principle. Alkaloids, flavonoids, coumarins, saponins and steroids are the compounds of plant origin known to have antibacterial activity. These compounds were detected in the SAE [40–44]. Hence further objective is to identify a potential lead compound, which can be developed into a candidate anti-infective drug, in particular for treatment of infections by multidrug resistant pathogens like MRSA and VRSA.

In the past few decades, MRSA have caused a major problem with nosocomial infections throughout the world [7]. In the developed countries, fluoroquinolones (ciprofloxacin and ofloxacin) are recommended for serious infections associated with Staphylococci[45] but vancomycin still remains the drug of choice for most MRSA-associated diseases [46]. In this study four MRSA isolates were resistant to vancomycin, five MRSA isolates were resistant and two were intermediately resistant to ciprofloxacin respectively, further emphasizing the difficulties in treatment of MRSA infections with antibiotics (Table 1). Moreover, in the last 2 decades, the number of new antimicrobial drugs that reached the marketplace has fallen to less than half the previous level [47]. Hence, more efforts should be directed towards the screening for new antimicrobial agents. The anti-MRSA activity exhibited by T.alternifolia stem bark extracts seems a promising step towards research for finding a new therapeutic agent against MRSA. However, extensive research needs to be carried out on this aspect of T. alternifolia.

The aqueous extract of T. alternifolia has antibacterial activity against MRSA and VRSA but does not retain any cytotoxicity, which further validates the use of the plant in traditional medicine. The present study lays the basis for future research, to validate the possible use of T. alternifolia as a candidate in the treatment of MRSA infections.