**5.1.1 Collection of plant material and preparation of methanol (MEOH) extract**

15 kg fresh whole plant of *A. paniculata* was procured from the botanical garden of Forest Research Institute of Malaysia (FRIM), Kuala Lumpur, Malaysia, during the month of April, 2009. The plant was identified by Dr. Richard Chung Cheng Kong (Ph.D., Taxonomist, FRIM). The voucher specimen (NMPC-Q25) has been deposited in the Herbarium, Faculty of Pharmacy, IIUM, Kuantan, Pahang DM, Malaysia for future references.

The fresh whole plant (15 kg) of *A. paniculata* was cleaned and dried in a protech laboratory air dryer (FDD-720-Malaysia) at 40°C for 7 days and pulverized to powdered form (5.6 kg, 37.33%) using Fritsch Universal Cutting Mill-PULVERISETTE 19-Germany. It was then stored in a desiccators at 2°C until further use. The air dried powder of whole plant (5 kg) of *A. paniculata* was extracted by macerating in double distilled methanol (20.0 L) at room temperature for 24 h, filtered, and evaporated under reduced pressure. The whole process was repeated thrice to ensure maximum yield of methanol soluble compounds from the plant powder. Each time, filtrate was evaporated under reduced pressure (Buchi Rotary Evaporator, R-210) and combined. The dark blackish green residue so obtained was further freeze dried to yield 305 g (6.1%) MeOH extract and was stored at 2°C in labeled sterile bottle until further antibacterial evaluation and isolation of antibacterial compounds.

### **5.1.2 Source of microorganism and preparation of standard bacterial suspensions**

*Staphylococcus aureus* (IMR S-277), *Streptococcus pyogenes* (IMR S-526)*, Micrococcus luteus* (IMR B-7), *Proteus mirabilis* (IMR P-74) and *Pseudomonas aeruginosa* (IMR P-84) were purchased from the Institute for Medical Research (IMR), Kuala Lumpur, Malaysia. The bacterial stock cultures were maintained on nutrient agar slants prior to use. The average number of viable, *S. aureus*, *S. pyogenes*, *M. luteus*, *P. mirabilis* and *P. aeruginosa* organisms per mL of the stock suspensions was determined by means of the surface viable counting technique (Omar, 1974). About 107-108 CFU/mL was used. Each time, a fresh stock suspension was prepared; the experimental conditions were maintained constant so that suspensions with very close viable counts could be obtained successfully.

#### **5.1.3** *In vitro* **antibacterial activity test for MeOH extract and determination of minimum inhibitory concentration (MIC)**

The cup-plate agar diffusion method was adopted according to Kokoska *et al*. (2002) to assess the antibacterial activity of the MeOH extract. 0.6 mL of standardized bacterial stock

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.

**5.1.1 Collection of plant material and preparation of methanol (MEOH) extract** 

of Pharmacy, IIUM, Kuantan, Pahang DM, Malaysia for future references.

15 kg fresh whole plant of *A. paniculata* was procured from the botanical garden of Forest Research Institute of Malaysia (FRIM), Kuala Lumpur, Malaysia, during the month of April, 2009. The plant was identified by Dr. Richard Chung Cheng Kong (Ph.D., Taxonomist, FRIM). The voucher specimen (NMPC-Q25) has been deposited in the Herbarium, Faculty

The fresh whole plant (15 kg) of *A. paniculata* was cleaned and dried in a protech laboratory air dryer (FDD-720-Malaysia) at 40°C for 7 days and pulverized to powdered form (5.6 kg, 37.33%) using Fritsch Universal Cutting Mill-PULVERISETTE 19-Germany. It was then stored in a desiccators at 2°C until further use. The air dried powder of whole plant (5 kg) of *A. paniculata* was extracted by macerating in double distilled methanol (20.0 L) at room temperature for 24 h, filtered, and evaporated under reduced pressure. The whole process was repeated thrice to ensure maximum yield of methanol soluble compounds from the plant powder. Each time, filtrate was evaporated under reduced pressure (Buchi Rotary Evaporator, R-210) and combined. The dark blackish green residue so obtained was further freeze dried to yield 305 g (6.1%) MeOH extract and was stored at 2°C in labeled sterile

bottle until further antibacterial evaluation and isolation of antibacterial compounds.

**5.1.3** *In vitro* **antibacterial activity test for MeOH extract and determination of** 

The cup-plate agar diffusion method was adopted according to Kokoska *et al*. (2002) to assess the antibacterial activity of the MeOH extract. 0.6 mL of standardized bacterial stock

**5.1.2 Source of microorganism and preparation of standard bacterial suspensions** 

*Staphylococcus aureus* (IMR S-277), *Streptococcus pyogenes* (IMR S-526)*, Micrococcus luteus* (IMR B-7), *Proteus mirabilis* (IMR P-74) and *Pseudomonas aeruginosa* (IMR P-84) were purchased from the Institute for Medical Research (IMR), Kuala Lumpur, Malaysia. The bacterial stock cultures were maintained on nutrient agar slants prior to use. The average number of viable, *S. aureus*, *S. pyogenes*, *M. luteus*, *P. mirabilis* and *P. aeruginosa* organisms per mL of the stock suspensions was determined by means of the surface viable counting technique (Omar, 1974). About 107-108 CFU/mL was used. Each time, a fresh stock suspension was prepared; the experimental conditions were maintained constant so that suspensions with very close

**5.1 Isolation of antibacterial compounds from** *A. paniculata* 

viable counts could be obtained successfully.

**minimum inhibitory concentration (MIC)** 

**5. Methods** 

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, averaged and the mean values were recorded.

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

#### **5.1.4 Bioassay guided isolation**

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

*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,

*Andrographis paniculata* (Burm.f) Wall. ex Ness: A Potent Antibacterial Plant 351

*14-deoxyandrographolide* (AB-2): M.P. 172-174°C, UV λmax MeOH nm: 223. IR (cm-1) ν: 3367, 1736, 1646, 896. 1H NMR (600 MHz, in DMSO-*d6*), δ (ppm): 1.32 (m, 2H, C1-CH2), 2.04 (brs, 1H, C2-CH2), 1.99 (o, 1H, C2-CH2), 3.35 (o, 1H, C3-CH-), 1.46 (o, 1H, C5-CH-), 2.18 (brs, 1H, C6-CH2), 1.98 (brd, J=6 Hz, 1H, C6-CH2), 2.45 (t, 2H, C7-CH2), 3.50 (o, 1H, C9-CH-), 2.43 (dd, J= 4.2, 2.4 Hz, 2H, C11-CH2), 2.55 (dd, J= 12, 7.2 Hz, 2H, C12-CH2), 7.10 (brs, 1H, C14-CH-), 4.91 (brs, 2H, C15-CH2), 4.59 (brs, 1H, C17-CH2), 4.45 (brs, 1H, C17-CH2), 1.59 (o, 3H, C18- CH3), 4.20 (brs, 1H, C19-CH2), 4.18 (brs, 1H, C19-CH2), 0.71 (s, 3H, C20-CH3), *"o" denotes overlapping signals*; 13C NMR (125.76 MHz, in DMSO*d*6), δ (ppm): 38.93 (C1), 37.74 (C2), 80.50 (C3), 64.13 (C4), 55.21 (C5), 28.22 (C6), 37.06 (C7), 148.94 (C8), 55.96 (C9), 42.96 (C10), 24.88 (C11), 23.76 (C12), 146.64 (C13), 127.83 (C14), 66.31 (C15), 172.17 (C16), 108.86 (C17), 22.67 (C18), 74.61 (C19), 15.16 (C20). From this spectral data and their direct comparison with the previously published data (Poonam *et al*., 2010) of the same compound, AB-2 was

The minimum inhibitory concentrations of the isolated compounds AB-1 and AB-2 were determined using the agar dilution method following the standard protocol of the European Committee for Antimicrobial Susceptibility Testing (EUCAST, 2003). The compounds were dissolved in 10% DMSO and 2-fold diluted in MHA to obtain 250, 125, 62.5, 31.3, 15.6 and 7.81 μgmL-1. The mixture of the media and compounds were thoroughly mixed and poured onto pre-labeled sterile Petri dishes on a level surface. Additional Petri dishes containing only the growth media were prepared in the same way so as to serve for comparison of growth of the respective bacteria. The plates were then set at room temperature and dried. The suspensions of the respective bacteria (corresponding to 108 CFU/mL) were inoculated onto the series of agar plates. The plates were then incubated at 37°C for 24 h. The experiments were performed in duplicate and MIC values expressed as the lowest concentration of the plant extracts that produced complete suppression of colony of

The experimental results were expressed as mean ± standard deviation (STD) of triplicate experiments. Statistical differences between the antibiotics and inhibition zones formed by the plant extract were detected by analysis of variance (ANOVA) using SPSS 19.0 statistical software (SPSS, Chicago, Illinois, USA) followed by the Tukey test for multiple comparisons between means. P values lower than 0.05 (p < 0.05) were considered significantly different

The results of the cup-plate agar diffusion method revealed that MeOH extract of *A. paniculata* whole plant extract do possess antibacterial activity against all 5 bacteria taken into consideration *in vitro* (Table 1). Maximum antibacterial activity was observed against *S. aureus* (19.67 ± 0.76 mm) at 1000 μgmL-1 and the least activity was detected against *P.* 

whereas P values lower than 0.01 (p < 0.01) were considered highly significant.

*aeruginosa* (7.00 ± 1.50 mm) at 250 μgmL-1, respectively.

unambiguously identified as 14-deoxyandrographolide (Fig. 4).

respective bacteria.

**6. Results** 

**5.1.7 Statistical analyses** 

**5.1.6 Minimum inhibitory concentration (MIC) of isolated compounds** 

allowing the chromatographic retention factors (R.f.) observation by viewing under UV light at 254 nm (short wave) and 366 nm (long wave) and comparing with the reference chromatogram (already sprayed with vanillin reagent and heated at 120°C) to note the antibacterial compounds. Vanillin reagent was prepared by dissolving 15 g of vanillin in ethanol (250 mL) and H2SO4 (2.5 mL). Vanillin reagent gives different colored spots with different compounds on TLC plate upon heating at 120°C (Rahalison *et al*., 2007).
