**3. Literature review on antibacterial studies of** *A. paniculata*

*A. paniculata* has been extensively used to treat a variety of conditions of infectious origin in traditional systems of medicine. Modern research has investigated it for antimicrobial activity against various pathogenic and non-pathogenic bacteria. For instance, Leelarasamee *et al*. (1990) reported that crude powder suspended in water had no *in vitro* antibacterial activity against *Salmonella, Shigella*, *Escherichia coli*, and *Staphylococcus aureus*, even at a concentration of

In this context, *Andrographis paniculata* (Burm.f.) Wall. ex Nees., could be a potential source to develop new efficacious antibacterial drugs. *A. paniculata* (*Acanthaceae*) (King of Bitters) is an annual herbaceous plant and is widely cultivated and traditionally used in Southern Asia, China and some parts of Europe. *A. paniculata* has been effectively used in traditional Asian medicines for centuries. In traditional medicine, *A. paniculata* is widely used to get rid of a body heat, dispel toxins from the body, prevents common cold, upper respiratory tract infections including sinusitis and fever (Gabrielian *et al*., 2002) and as an antidote against snakes and insects poisons (Samy *et al*., 2008)*. A. paniculata* has been reported to exhibit various mode of biological activities *in vivo* as well as *in vitro* viz., antiviral (Wiart *et al*., 2000), anti-inflammatory (Wen *et al*., 2010), antihuman immunodeficiency virus (HIV) (Calabrese *et al*., 2000), immunomodulating/immunostimulatory (Iruretagoyena *et al*., 2005), anticancer activity (Li *et al*., 2007; Geethangili *et al*., 2008) and antibacterial activity (Leelarasamee *et al*., 1990; Singha *et al*., 2003; Zaidan *et al*., 2005; Xu *et al*., 2006; Voravuthikunchai *et al*., 2006; Mishra *et al*., 2009; Sahalan *et al*., 2010; Abubacker and Vasanth, 2010; Kataky and Handique, 2010; Parvataneni and Koduru, 2010; Roy *et al*., 2010;

The characteristic secondary metabolites encountered in *A. paniculata* have considerably enhanced its importance in the arena of medicinal plants. It is specifically rated high in therapeutic action in curing liver disorders, common cough and colds in human (Niranjan *et al*., 2010). *A. paniculata* chiefly contains diterpenes, lactones, and flavonoids. Flavonoids mainly exist in the root, but have also been isolated from the leaves. The aerial parts contain alkanes, ketones, and aldehydes. Although it was initially thought that the bitter substance in the leaves was the lactone andrographolide, later investigations revealed that the leaves contained two bitter principles-andrographolide and a compound named kalmeghin. Four lactoneschuanxinlian A (deoxyandrographolide), B (andrographolide), C (neoandrographolide) and D (14-deoxy-11,12-didehydroandrographolide)-were isolated from the aerial parts in China (Chang and But, 1987). A diterpene glucoside (deoxyandrographolide-19-β-D-glucoside) has been detected in the leaves (Weiming *et al*., 1982) and six diterpenoids of the ent-labdane type, two diterpene glucosides and four diterpene dimers (bis-andrographolides A, B, C, and D) have been isolated from aerial parts (Matsuda *et al*., 1994). Two flavonoids identified as 5,7,2',3' tetramethoxyflavanone and 5-hydroxy-7,2',3'-trimethoxyflavone were isolated from the whole plant (Koteswara *et al*., 2004), while 12 new flavonoids and 14 diterpenoids have been reported from the aerial parts (Chen *et al*., 2006a, 2006b). Two new flavonoid glycosides and a new diterpenoid (andrographic acid) were recently reported (Li *et al*., 2007), and two new entlabdane diterpenoid glycosides were also isolated from the aerial parts of *A. paniculata* (Zhou *et* 

Sule *et al*., 2011a, 2011b).

*al*., 2008).

**2. Phytochemical investigations of** *A. paniculata*

**3. Literature review on antibacterial studies of** *A. paniculata* 

*A. paniculata* has been extensively used to treat a variety of conditions of infectious origin in traditional systems of medicine. Modern research has investigated it for antimicrobial activity against various pathogenic and non-pathogenic bacteria. For instance, Leelarasamee *et al*. (1990) reported that crude powder suspended in water had no *in vitro* antibacterial activity against *Salmonella, Shigella*, *Escherichia coli*, and *Staphylococcus aureus*, even at a concentration of 25 mg/mL crude powder. Moreover, administration of a single oral dose of powder, up to 6 g, to healthy volunteers in a randomized crossover manner or daily administration of 0.12-24 g/kg body weight to rats for six months also failed to show any *ex vivo* antibacterial activity. A similar conclusion was also reached by Zaidan *et al.* (2005) who found crude aqueous extract of leaves had no activity against *Escherichia coli* or *Klebsiella pneumoniae* but exhibited significant antimicrobial activity against gram positive *S. aureus*, methicillin-resistant *S. aureus* (MRSA), and gram-negative *Pseudomonas aeruginosa*. However, Singha *et al*. (2003) reported significant antibacterial activity of an aqueous extract and attributed it to the combined effect of andrographolides and arabinogalactan proteins. In contrast, Xu *et al*. (2006) investigated the antimicrobial activity using *A. paniculata* methanolic and aqueous extracts and authentic andrographolide against nine human bacterial pathogens. Their results indicated methanolic extracts of *A. paniculata* to be active against only two of the pathogens, while authentic andrographolide did not show any activity. They concluded that the observed antimicrobial activity was due to other active principle(s) present in the extracts that were used in the investigation. The ethanol extract was also reported to be devoid of significant antibacterial activity against enterohemorrhagic strains of *E. coli* (Voravuthikunchai *et al*., 2006). In another study, Sahalan *et al*. (2010) reported the antibacterial activity of methanol extract of the leaves of *A. paniculata* against *Staphylococcus aureus*, *Bacillus subtilis*, *Streptococcus epidemidis*, *Escherichia coli*, *Klebsiella pneumoniae* and *Pseudomonas aeruginosa*. Abubacker and Vasanth (2010) reported the antibacterial value of ethanol leaf extract against pathogenic bacteria *Escherichia coli, Klebsiella peneumoniae, Proteus vulgaris* and *Streptococcus pneumonia.* Bioactive compound andrographolide was isolated from the leaf. The results revealed that the ethanol leaf extract and andrographolide compound are potent in inhibiting these bacteria and this work highlights that the inhibitory effect is on par with standard antibiotics. Kataky and Handique (2010) reported antimicrobial activity of various organic and aqueous extracts of eight-months old micropropagated plantlets of *A. paniculata* against gram negative (*Klebsiella pneumoniae*, *Escherichia coli*, *Pseudomonas aeruginosa*), gram positive (*Staphylococcus aureus* and *Bacillus subtilis*) bacteria. Among all tested extracts, chloroform extract showed strong inhibitory activity with all the microbes tested. Out of the five microbial test organisms *Staphylococcus aureus* was the most susceptible. The minimal inhibitory concentration (MIC) of the chloroform extract ranged from 15.625 μg/mL to 31.5 μg/mL. Roy *et al*. (2010) reported the antibacterial potential of chloroform extract of the aerial parts of *A. paniculata* against *E. faecalis* (35 mm), followed by *E. cloacae* (30 mm) *P. aeruginosa* (28 mm) and *E. coli* (25 mm). Least inhibition zone was observed against *S. aureus* (15 mm). Though the inhibition zone observed against *S. typhimurium* was only 18 mm, it is noteworthy when comparing it with that of the control result. Out of the 9 pathogenic strains tested, 7 strains showed inhibition zones comparable with that of the control (amikacin) used. The chloroform extract antimicrobial activity seen against all the tested gram-negative opportunistic and pathogenic bacteria is very encouraging and important considering the role of gram-negative bacteria in noscomial infections leading to increased morbidity and mortality rates. Sule *et al*. (2011a) reported that *A. paniculata* extracts have bactericidal characteristic against most of the Gram positive bacteria and bacteriostatic activity against both Gram negative and Gram positive bacteria.

#### **4. Objective of current study**

*A. paniculata* has already been reported for its significant antibacterial potential by many researchers across the world (Leelarasamee *et al*., 1990; Singha *et al*., 2003; Zaidan *et al*., 2005;

*Andrographis paniculata* (Burm.f) Wall. ex Ness: A Potent Antibacterial Plant 349

suspensions corresponding to 107-108 CFU/mL was thoroughly mixed with 60 mL of sterile nutrient agar. 20 mL of the inoculated nutrient agar were distributed into sterile labeled Petri dishes. The agar was left to set at room temperature and in each of these plates, 3 cups 6 mm in diameter were punched using a sterile cork borer allowing at least 30 mm between adjacent wells and the agar discs were removed. Fixed volumes of the plant extract (1000, 500 and 250 μgmL-1) were then introduced into each wells using micro titer-pipette and allowed to diffuse at room temperature for two hours. In separate wells, 30 µg each of gentamicin and vancomycin were added as positive controls whereas 10% DMSO was taken as negative control. The plates were then incubated in the upright position at 37°C for 24 h. Three replicates were carried out for the extract against each of the test organism. After incubation the diameter of the results and growth inhibition zones were measured,

Micro broth dilution method was used for the determination of MIC values for each plant extract showing antibacterial activity against test pathogens (NCCLS, 2003). Serial dilutions of the extracts were carried out in 10% DMSO (which had no inhibitory activity against test microorganisms) to make 500 μgmL-1 final concentration, this was then two fold serially diluted by adding to the broth media in a 96-wells micro titer plates to obtain 250, 125, 62.5, 31.3, 15.6 and 7.81 μgmL-1. Thereafter, 100 μL inoculum (108 CFU/mL) was added to each well. Bacterial suspensions were used as negative control, while broth containing standard drug (vancomycin and gentamicin) were used separately as positive controls. The micro titer plates were incubated at 37°C for 24 h. Each extract was assayed in duplicate; one was kept for incubation while the other was kept at 4°C for comparing the turbidity in the wells of micro plate. The MIC values were taken as the lowest concentration of the extracts in the well of the micro titer plate that showed no turbidity after incubation. The turbidity of the wells in the micro titer plate was interpreted as visible growth of microorganisms. Antibacterial index (AbI) of MeOH whole plant extract of *A. paniculata* was calculated separately as the average value of zone of inhibition against the Gram-positive and Gram-

To sterilized 8 x 4 cm silica gel 60 F254 TLC plates (Merck, Germany), 10 μL of MeOH extract was applied as small spots and the plates were developed in hexane:acetone (2:1) in duplicate (a TLC plate was used as the bioautogram while the other served as a chromatogram for reference in comparison with the bioautograph). The TLC plates were dried in an oven at 25°C for 7 h to activate the plates by absorbing the moisture content from

*S. aureus* and *P. mirabilis* were used as the indicator microorganisms for the bioautography of antibacterial compounds from the MeOH extract of *A. paniculata*. 200 μL each from broth cultures of *S. aureus* and *P. mirabilis* (adjusted to 108 CFU/mL) were mixed with 35 mL molten Mueller-Hinton agar (MHA) at 30°C separately. The suspensions of agar and bacteria were spread aseptically onto the already developed TLC plates in square Petri dishes (8 x 4 cm), allowed for 30 mins to solidify and the plates were incubated at 37°C for 24 h. At the end of incubation time, 0.5% *p*-Iodonitrotetrazolium Violet (INT) was sprayed on the plates for 5 mins. The active antibacterial compounds in the plant extracts formed clear zones of inhibition on the TLC plates against a deep pink back ground of bacterial growth,

averaged and the mean values were recorded.

negative bacteria, respectively (Zakaria *et al*., 2007).

the plates and removing all residual solvents (Veronica *et al*., 2005).

**5.1.4 Bioassay guided isolation** 

Xu *et al*., 2006; Voravuthikunchai *et al*., 2006; Mishra *et al*., 2009; Sahalan *et al*., 2010; Abubacker and Vasanth, 2010; Kataky and Handique, 2010; Parvataneni and Koduru, 2010; Roy *et al*., 2010; Sule *et al*., 2011a, 2011b). However, no attempt has ever been made to identify and isolate active principles responsible for unleashing its true antibacterial activity. Identification and isolation of active principles from *A. paniculata* might prove promising antibacterial agents through foreseeable future endeavors. Hence, this study was a scrupulous attempt to identify and isolate pure antibacterial compounds from the methanol extract of the whole plant of *A. paniculata* through bioassay guided isolation method.
