**6. Results**

350 Antimicrobial Agents

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).

the same compounds.

**5.1.5 Identification and isolation of antibacterial compounds from MeOH extract** 

100 g MeOH extract was loaded onto column (10 x 50 cm) packed with silica gel 60 particle size 0.063-0.2 mm (70-230 mesh) (Fluka Chemika) as stationery phase, the extracts as the mobile phase and the column was eluted with ten different concentrations of hexane:ethylacetate (9:1-1:9) and finally with ethylacetate:methanol (9:1-1:9) solvent systems (with gradual increase in polarity), 190 fractions were obtained and pooled to give a total of 20 (M1-M20) similar fractions based on their R.f. values as indicated by TLC plate analysis in chloroform:methanol:ethylacetate (CHCl3:MeOH:EtOAc; 16:0.8:1.2) solvent system. Antibacterial active fractions M9-M13 and M16-M18 as indicated by bioautography of the extract afforded two crystallized compounds which were further purified by using preparative column chromatography on silica gel 60 and eluted with hexane:ethylacetate (9:1-1:9) to produce 76 (P1- P76) fractions. Eluents in test tubes P1- P10, P12- P21, and P22- P35 upon crystallization with absolute ethanol afforded pure antibacterial compound AB-1 (white crystals, 29 mg, R.f. 0.70-CHCl3:MeOH:EtOAc, 16: 0.8: 1.2). Fractions P41- P58 upon crystallization with absolute ethanol afforded pure antibacterial compound AB-2 (colourless crystals, 35 mg, R.f. 0.64 (CHCl3:MeOH:EtOAc; 16: 0.8: 1.2), respectively. The purity of both antibacterial compounds were further determined through high performance liquid chromatography (HPLC) and running TLC plates in different binary and ternary solvent systems. Structures of both antibacterial compounds were elucidated through the integration of 1H- and 13C NMR spectra and comparison of their physical, chemical and spectral data was made with the previous reported data of

*3-O-β-D-glucosyl-14-deoxyandrographolides* (AB-1): M.P. 242-244°C, UV λmax MeOH nm: 202. IR (cm-1) ν: 3351, 1732, 165, 899. 1H NMR (600 MHz, in DMSO-*d6*), δ (ppm): 1.25 (o, 1H, C1- CH2), 1.71 (o, 1H, C1-CH2), 2.10 (m, 1H, C2-CH2), 1.98 (m, 1H, C2-CH2), 3.92 (o, 1H, C3-CH- ), 1.3 (m, 1H, C5-CH-), 1.85 (m, 2H, C6-CH2), 2.4 (m, 2H, C7-CH2), 3.35 (d, J=8.4 Hz, 1H, C9- CH-), 1.8 (m, 2H, C11-CH2), 2.6 (m, 1H, C12-CH2), 2.3 (m, 1H, C12-CH2), 7.10 (t, 1H, C14- CH-), 4.77 (brs, 2H, C15-CH2), 4.87 (brs, 1H, C17-CH2), 4.59 (brs, 1H, C17-CH2), 1.007 (brs, 3H, C18-CH3), 4.03 (d, J=9.6 Hz, 1H, C19-CH2), 3.22 (d, J=9.6, 1H, C19-CH2), 0.66 (brs, 3H, C20-CH3), 4.24 (d, J=7.2 Hz, 1H, C1'-CH-), 3.37 (o, 1H, C2'-CH-), 3.39 (o, 1H, C3'-CH-), 3.57 (o, 1H, C4'-CH-), 3.59 (o, 1H, C5'-CH-), 3.84 (dd, J=11.4, 4.8 Hz, 2H, C6'-CH2), *"o" denotes overlapping signals*; 13C NMR (125.76 MHz, in DMSO*d*6), δ (ppm): 38.19 (C1), 29.72 (C2), 75.04 (C3), 39.56 (C4), 56.44 (C5), 35.97 (C6), 38.44 (C7), 147.30 (C8), 56.18 (C9), 38.89 (C10), 21.72 (C11), 24.46 (C12), 136.02 (C13), 143.84 (C14), 70.11 (C15), 174.33 (C16), 107.08 (C17), 18.93 (C18), 62.61 (C19), 15.41 (C20), 103.10 (C1'), 71.72 (C2'), 72.73 (C3'), 74.03 (C4'), 76.23 (C5'), 70.72 (C6'). From this spectral data and their direct comparison with the previously published spectral data (Zhou *et al*., 2008) of the same compound, AB-1 was unambiguously

identified as 3-*O*-β-D-glucosyl-14-deoxyandrographolide (Fig. 3).

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

*Andrographis paniculata* (Burm.f) Wall. ex Ness: A Potent Antibacterial Plant 353

(a) (b) Fig. 1. Bioautography of AB-1 against *S. aureus*. (a) Referenced chromatogram sprayed with

(a) (b)

Fig. 2. Bioautography of AB-2 against *S. aureus*. (a) Bioautogram against *S. aureus*. (b)

Referenced chromatogram viewed under UV light.

vanillin/H2SO4 spray reagent. (b) Bioautogram against *S. aureus*.


Comparison with antibiotics \*\*P < 0.01 highly significant \*P < 0.05 significant difference - No Activity

Table 1. Antibacterial activity of MeOH extract of the whole plant of *A. paniculata*. Numbers indicate the mean diameters of inhibition of triplicate experiments ± standard deviation (SD)

Bioautography of MeOH extract revealed two prominent spots on the bioautogram against *S. aureus* and *P. mirabilis* which were used as an indicator organism and consequently led to the identication and subsequent isolation of two antibacterial compounds viz., an entlabdane diterpene glycoside (AB-1) and a diterpene lactone (AB-2) as the main active principles. Both compounds were active against *S. aureus* (Fig. 1 & 2) and *P. mirabilis* which were used as indicator organisms in the bioautography technique on TLC plates forming clear zones against pink background of the living microorganisms when compared to the reference chromatogram.

**Zones of Inhibition (mm) Bacterial strains Plant Extract 1000 µg/ml 500 µg/ml 250 µg/ml Antibiotics** 

(IMR S-277) MeOH 19.67 ± 0.76\* 18.00 ± 0.50 14.00 ±1.00\*\* 17.00 ± 1.05

(IMR B-7) MeOH 18.50 ± 0.58 15.00 ± 0.89\* 13.00 ±0.58\*\* 19.00 ± 0.50

(IMR S-526) MeOH 16.00 ± 0.58\* 13.00 ± 0.74 10.67 ±1.15\*\* 14.50 ± 1.00

IMR P-76 MeOH 14.50±0.58\*\* 14.00±0.58\*\* 10.50 ±1.00\*\* 21.33 ± 0.89

IMR P-84 MeOH 11.50±0.50\*\* 9.00±1.26\*\* 7.00±1.50\*\* 18.50 ± 0.50

Table 1. Antibacterial activity of MeOH extract of the whole plant of *A. paniculata*. Numbers indicate the mean diameters of inhibition of triplicate experiments ± standard deviation (SD)

Bioautography of MeOH extract revealed two prominent spots on the bioautogram against *S. aureus* and *P. mirabilis* which were used as an indicator organism and consequently led to the identication and subsequent isolation of two antibacterial compounds viz., an entlabdane diterpene glycoside (AB-1) and a diterpene lactone (AB-2) as the main active principles. Both compounds were active against *S. aureus* (Fig. 1 & 2) and *P. mirabilis* which were used as indicator organisms in the bioautography technique on TLC plates forming clear zones against pink background of the living microorganisms when compared to the

*S. aureus* 

*M. luteus* 

*S. pyogenes* 

*P. mirabilis* 

*P. aeruginosa* 


Comparison with antibiotics \*\*P < 0.01 highly significant \*P < 0.05 significant difference

reference chromatogram.

**Gram Positive Strains Vancomycin** 

**Gram Negative Strains Gentamicin** 

**(30 µg)** 

Fig. 1. Bioautography of AB-1 against *S. aureus*. (a) Referenced chromatogram sprayed with vanillin/H2SO4 spray reagent. (b) Bioautogram against *S. aureus*.

(a) (b)

(a) (b)

Fig. 2. Bioautography of AB-2 against *S. aureus*. (a) Bioautogram against *S. aureus*. (b) Referenced chromatogram viewed under UV light.

*Andrographis paniculata* (Burm.f) Wall. ex Ness: A Potent Antibacterial Plant 355

72.73, 74.03, 76.23, and 70.72 and the characteristic signals for the double bond containing one hydrogen at carbon 14 in γ-lactone ring were observed at δ 7.10 (t, 1H) in 1HNMR as well as in 13C at δ 143.84, respectively, which corresponds to the 3-*O*-β-D-glucosyl-14-

O

Fig. 3. Structure of AB-1 (3-*O*-β-D-glucosyl-14-deoxyandrographolide) based on 1H- and 13C

AB-2 was also found to be positive for the Legal and Kedde reactions, suggesting the presence of an α, β-unsaturated lactone in the molecule. The characteristic NMR spectral data indicated that compound AB-2 was a labdane-type diterpene with α, β-unsaturated γ-lactone. In the 1H-NMR spectrum of AB-2, two methyl singlets were observed at δ 0.71 and 1.59, respectively. The characteristic exocyclic methylene protons for AB-2 diterpenoids were observed at δ 4.59 (brs, 1H) and 4.45 (brs, 1H) in 1HNMR as well as at δ 108.86 in 13C, respectively. The 1H- and 13C-NMR (in CDCl3) spectra of AB-2 suggested a diterpenoid compound with a structure

CH3

H3C H2COH

Fig. 4. Structure of AB-2 (14-deoxyandrographolide) based on 1H- and 13C NMR spectra.

H

CH2

CH2OH

O

O

CH2

O

O

H

similar to that of 14-deoxyandrographolide (Poonam *et al*., 2010) (Fig. 4).

O

OH

HO

deoxyandrographolide (Zhou *et al*., 2008) (Fig. 3).

HO

OH

HO

NMR spectra

Minimum inhibitory concentration (MIC) values for MeOH extract and isolated compounds are shown in Table 2. MIC of MeOH extract ranged from 125-250 μgmL-1 with the highest MIC value exerted by the extract against *S. pyogenes, P. mirabilis* and *P. aeruginosa* (250 μgmL-1) and the least against *S. aureus* and *M. luteus* (125 μgmL-1). MIC values for both isolated compounds ranged from 15.6-250 μgmL-1. Highest MIC value was exerted by compound AB-1 against *P. aeruginosa* (250 μgmL-1) while the least was exerted by compound AB-2 against *S. aureus* (15.6 μgmL-1), however, no activity was exerted by compound AB-1 against *M. luteus* (Table 2)*.* The MeOH extract's antibacterial index (AbI) was best against Grampositive strains tested as compared to the Gram-negative strains with mean inhibition zones of 13.9 mm and 10.4 mm, respectively (Table 3).


\*NA-no activity

Table 2. Minimum inhibitory concentrations (MIC) of the MeOH extract of the whole plant of *A. paniculata* and isolated compounds against bacterial strains


Table 3. Antibacterial activity indexes (AbI) of MeOH extract of the whole plant of *A. paniculata* 

Compound AB-1 gave positive Legal and Kedde test, suggesting the presence of an α, βunsaturated lactone in the compound. The 1H- and 13C-NMR spectra of AB-1 revealed signals due to a β-glucopyranosyl group [δH 4.24 (d, J=7.2 Hz, 1H)] and δC 103.10, 71.72,

Minimum inhibitory concentration (MIC) values for MeOH extract and isolated compounds are shown in Table 2. MIC of MeOH extract ranged from 125-250 μgmL-1 with the highest MIC value exerted by the extract against *S. pyogenes, P. mirabilis* and *P. aeruginosa* (250 μgmL-1) and the least against *S. aureus* and *M. luteus* (125 μgmL-1). MIC values for both isolated compounds ranged from 15.6-250 μgmL-1. Highest MIC value was exerted by compound AB-1 against *P. aeruginosa* (250 μgmL-1) while the least was exerted by compound AB-2 against *S. aureus* (15.6 μgmL-1), however, no activity was exerted by compound AB-1 against *M. luteus* (Table 2)*.* The MeOH extract's antibacterial index (AbI) was best against Grampositive strains tested as compared to the Gram-negative strains with mean inhibition zones

**MeOH Compounds** 

**Extract AB-1 AB-2** 

**Activity Index (mm)** 

**MIC (µg/ml)** 

**Gram Negative** 

of *A. paniculata* and isolated compounds against bacterial strains

**Strains MeOH Extract** 

*P. mirabilis, P. aeruginosa* 10.4

*S. aureus* 125 62.5 15.6 *M. luteus* 125 N/A 125 *S. pyogenes* 250 125 62.5

*P. mirabilis* 250 125 125 *P. aeruginosa* 250 250 250

Table 2. Minimum inhibitory concentrations (MIC) of the MeOH extract of the whole plant

Table 3. Antibacterial activity indexes (AbI) of MeOH extract of the whole plant of *A.* 

Compound AB-1 gave positive Legal and Kedde test, suggesting the presence of an α, βunsaturated lactone in the compound. The 1H- and 13C-NMR spectra of AB-1 revealed signals due to a β-glucopyranosyl group [δH 4.24 (d, J=7.2 Hz, 1H)] and δC 103.10, 71.72,

13.9

of 13.9 mm and 10.4 mm, respectively (Table 3).

**Gram Positive** 

Gram Positive *S. aureus, M. luteus, S.* 

Gram Negative

*pyogenes*

\*NA-no activity

*paniculata* 

72.73, 74.03, 76.23, and 70.72 and the characteristic signals for the double bond containing one hydrogen at carbon 14 in γ-lactone ring were observed at δ 7.10 (t, 1H) in 1HNMR as well as in 13C at δ 143.84, respectively, which corresponds to the 3-*O*-β-D-glucosyl-14 deoxyandrographolide (Zhou *et al*., 2008) (Fig. 3).

Fig. 3. Structure of AB-1 (3-*O*-β-D-glucosyl-14-deoxyandrographolide) based on 1H- and 13C NMR spectra

AB-2 was also found to be positive for the Legal and Kedde reactions, suggesting the presence of an α, β-unsaturated lactone in the molecule. The characteristic NMR spectral data indicated that compound AB-2 was a labdane-type diterpene with α, β-unsaturated γ-lactone. In the 1H-NMR spectrum of AB-2, two methyl singlets were observed at δ 0.71 and 1.59, respectively. The characteristic exocyclic methylene protons for AB-2 diterpenoids were observed at δ 4.59 (brs, 1H) and 4.45 (brs, 1H) in 1HNMR as well as at δ 108.86 in 13C, respectively. The 1H- and 13C-NMR (in CDCl3) spectra of AB-2 suggested a diterpenoid compound with a structure similar to that of 14-deoxyandrographolide (Poonam *et al*., 2010) (Fig. 4).

Fig. 4. Structure of AB-2 (14-deoxyandrographolide) based on 1H- and 13C NMR spectra.

*Andrographis paniculata* (Burm.f) Wall. ex Ness: A Potent Antibacterial Plant 357

an effective permeability barrier. The cell walls of Gram-negative organisms are more complex in lay out than the Gram-positive ones acting as a diffusion barrier and making them less susceptible to the antimicrobial agents than are Gram-positive bacteria (Nikaido, 2003). In the present study, after the first chromatography of the MeOH extract of the whole plant of *A. paniculata* on a silica gel column, the antibacterial activity of the collected fractions were tested against *S. aureus* and *P. mirabilis* using bio-autography on a TLC plate. This revealed that all the fractions except nine were very active against *S. aureus* and *P. mirabilis*. Isolation of these compounds in pure form was achieved by repeated washing of the crystalline matter off the green coloring material with toluene and repeated recrystallization with absolute ethanol and final washing of the crystals with cold methanol. The purity of the sample at every stage of recrystallization was monitored through TLC.

The thin layer chromatography bioautography-guided strategy was successfully used to isolate two antibacterial compounds from MeOH extract of *A. paniculata* whole plant for the first time. The 3-*O*-β-D-glycosyl-14-deoxyandrographolide and 14-deoxyandrographolide demonstrated signicant antibacterial activities against the selected microbial strains. Quantitative HPLC and TLC analysis conrmed that these isolated compounds are predominate components in whole plant MeOH extract, indicating their signicant contribution to the overall antibacterial activity. Further investigation of the activities of these compounds and their potential use in the treatment of bacterial diseases are still

Abubacker, M.N., S. Vasantha, 2010. Antibacterial activity of ethanolic leaf extract of

Bächi, B.B., 2002. Resistance mechanisms of Gram-positive bacteria. *J Med Microb*., 292: 27-35. Calabrese, C., S.H. Berman, J.G. Babish, M. Xinfang, L. Shinto, M. Dorr, K. Wells, C.A.

Chang, H.M., P.P.H. But, 1987. *Pharmacology and Applications of Chinese Materia Medica*.

Hong Kong), Singapore: World Scientific Publishing Co. Pte. Ltd; 2: 918-928. Chen, L.X., G.X. Qu, F. Qiu, 2006a. Studies on diterpenoids from *Andrographis paniculata*.

Chen, L.X., G.X. Qu, F. Qiu, 2006b. Studies on flavonoids of *Andrographis paniculata*.

Coon, J.T., E. Ernst, 2004. *Andrographis paniculata* in the treatment of upper respiratory tract infections: a systematic review of safety and efficacy. *Planta Med*., 70: 293-298. European Committee on Antimicrobial Susceptibility Testing (EUCAST), 2003. Discussion

andrographolide. *Drug Invention Today* 2: 440-442.

*Zhongguo Zhong Yao Za Zhi* 31: 1594-1597.

*Zhongguo Zhong Yao Za Zhi* 31: 391-395.

patients and normal volunteers. *Phytotherapy Res*., 14: 333-338.

antibacterial agents by broth dilution. *Clin Microb Infect*., 9: 1-7.

*Andrographis paniculata* Nees (Acanthaceae) and its bioactive compound

Wenner, L.J. Standish, 2000. A phase I trial of andrographolide in HIV positive

English translation by Shem Chang-Shing Yeung, Sih Cheng-Yao and Lai-Ling Wang (Chinese Medicinal Material Research Centre, The Chinese University of

document, determination of minimum inhibitory concentrations (MICs) of

**8. Conclusion** 

sought.

**9. References** 

### **7. Discussion**

Antibiotics offer the core basis for the effective therapy of chronic bacterial infections. However, the high genetic variability of bacteria enables them to rapidly elude the action of antibiotics by developing antibiotic resistance. As resistance becomes more common, there becomes a greater need for alternative treatments. However, despite a push for new antibiotic therapies, there has been a continued decline in the number of newly approved drugs (Bächi, 2002). According to the World Health Organization (WHO) report on infectious diseases in 2000, overcoming antibiotic resistance is the major issue of the WHO for the next millennium. Hence, the last decade witnessed an increase in the investigations on plants as a source of human disease management (Paul *et al*., 2006). *A. paniculata* is common throughout Southeast Asia and India and is extensively used by traditional healers for the treatment of a wide variety of ailments (Coon and Ernst, 2004). The antibacterial activity of *A. paniculata* extracts are well known (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; Sule *et al*., 2011a, 2011b). Whilst many studies have isolated and characterized *A. paniculata* compounds, no study has ever determined the antimicrobial activity of isolated compounds so far. In the present experiment, the MeOH extract of the whole plant of *A. paniculata* showed broad spectrum antibacterial activity. 3-*O*-β-D-glycosyl-14-deoxyandrographolide and 14-deoxyandrographolide were isolated as active principles, which may serve as lead for the development of new pharmaceuticals that might address the unmet therapeutic needs to cure chronic bacterial infections effectively. The obvious fields where the natural product chemist can harvest benefits from a cooperation with the microbiologists are development of bioassay for efficient monitoring of isolation and purification of new compounds; bioassay fingerprinting to help early de-selection of known compounds (hereby supplementing the chemical data and giving additional avenues for tapping into the computerized data bases); activity spectrum to help de-selecting the very toxic compounds; obtaining a sharper focus in the natural product chemistry work on biologically active compounds. Novel and potentially useful may be of more interest than to go exclusively for just novelty (Lene, 1996). Bio-autography provides more information about plant compounds requires a smaller weight of sample and can be used for the bioassay-guided isolation of biological active compounds, simplifying the process of the identification and isolation of the active compounds (Rahalison *et al*., 2007).

The antibacterial activity measured by the cup-plate agar diffusion method was more prominent on the Gram-positive bacteria (*S. aureus, M. luteus* and *S. pyogenes*) than the Gram-negative bacteria (*P. mirabilis* and *P. aeruginosa*). Gram-positive bacteria were the most susceptible to growth inhibition by MeOH extract of *A. paniculata* whole plant. The greater susceptibility of Gram-positive bacteria has been previously reported for South American (Paz *et al*., 1995), African (Kudi *et al*., 1999) and Australian (Palombo and Semple, 2001) plant extracts. Susceptibility differences between Gram-positive and Gram-negative bacteria may be due to cell wall structural differences between these classes of bacteria. Gram-negative bacteria have an outer phospholipid membrane carrying the structural lipopolysaccharide components. This makes the cell wall impermeable to antimicrobial chemical substances. The Gram-positive bacteria tested were more susceptible to the plant extracts because it is well known that all Gram-positive bacteria have an outer peptidoglycan layer which is not an effective permeability barrier. The cell walls of Gram-negative organisms are more complex in lay out than the Gram-positive ones acting as a diffusion barrier and making them less susceptible to the antimicrobial agents than are Gram-positive bacteria (Nikaido, 2003). In the present study, after the first chromatography of the MeOH extract of the whole plant of *A. paniculata* on a silica gel column, the antibacterial activity of the collected fractions were tested against *S. aureus* and *P. mirabilis* using bio-autography on a TLC plate. This revealed that all the fractions except nine were very active against *S. aureus* and *P. mirabilis*. Isolation of these compounds in pure form was achieved by repeated washing of the crystalline matter off the green coloring material with toluene and repeated recrystallization with absolute ethanol and final washing of the crystals with cold methanol. The purity of the sample at every stage of recrystallization was monitored through TLC.
