A new pentacyclic triterpene with potent antibacterial activity from Limnophila indica Linn. (Druce)

Fitoterapia 90 (2013) 104–111

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A new pentacyclic triterpene with potent antibacterial activity from Limnophila indica Linn. (Druce) Goutam Brahmachari a,⁎, Narayan C. Mandal b, Rajiv Roy a, Ranjan Ghosh b, Soma Barman b, Sajal Sarkar a, Shyamal K. Jash a, Sadhan Mondal a a b

Laboratory of Natural Products and Organic Synthesis, Department of Chemistry, Visva-Bharati University, Santiniketan 731 235, West Bengal, India Microbiology and Plant Pathology Laboratory, Department of Botany, Visva-Bharati University, Santiniketan 731 235, West Bengal, India

a r t i c l e

i n f o

Article history: Received 6 February 2013 Accepted in revised form 10 July 2013 Available online 20 July 2013 Keywords: Limnophila indica Scrophulariaceae Pentacyclic triterpene 3-Oxo-olean-12(13), 18(19)-dien-29α-carboxylic acid Antibacterial activity

a b s t r a c t A new pentacyclic triterpenoid constituent, characterized as 3-oxo-olean-12(13),18(19)dien-29α-carboxylic acid (1) on the basis of detailed spectral studies, was isolated from the aerial parts and roots of Limnophila indica (Scrophulariaceae). Compound 1 exhibited considerable antibacterial activity against three Gram-positive bacteria viz. Bacillus subtilis, Staphylococcus aureus and Listeria monocytogenes (MICs within a range of 25–30 μg/ml) and moderate activity against four Gram-negative bacteria Salmonella typhimurium, Escherichia coli, Pseudomonas aeruginosa, and Pantoea ananatis (MICs within a range of 30–100 μg/ml). The plant pathogenic bacterium P. ananatis and human pathogenic S. typhimurium responded at comparatively higher concentrations of the compound 1, which were 75 and 100 μg/ml respectively. The compound inhibited the growth of Gram-positive B. subtilis and Gramnegative P. aeruginosa completely with a clear bactericidal mode of action at their MIC values. The compound upon treatment on both B. subtilis and P. aeruginosa released substantial amount of nucleic acid in the external medium and also effected the change of morphology towards pleomorphicity, thereby indicating its probable action on cell membrane. Furthermore, the triterpenoid 1 was found not to inhibit a probiotic lactic acid bacterium Lactococcus lactis subsp. lactis LABW4 under in vitro condition and to possess no toxicity in Swiss albino mice. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Limnophila (family: Scrophulariaceae) [1–6] is originated from a Latin word that means pond-loving indicating its existence in aquatic environments. It is commonly known as

Abbreviations: CC, column chromatography; CFU, colony forming units; CLSI, Clinical and Laboratory Standards Institute; DMSO, dimethyl sulphoxide; GCMS, gas chromatographic mass spectrophotometer; MIC, minimum inhibitory concentration; MTCC, microbial type culture collection; OA, oleanolic acid; SEM, scanning electron microscopy; SGOT, serum glutamic-oxaloacetic transaminase; SGPT, serum glutamic pyruvic transaminase ⁎ Corresponding author. Tel./fax: +91 3463 261526. E-mail addresses: [email protected], [email protected] (G. Brahmachari). 0367-326X/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.fitote.2013.07.012

‘Ambulia’ (Asian marshweed). It is a perennial from Southeast Asia, tropical to subtropical Africa, Australia, and Pacific Islands; also finds adventive distribution in North America. Limnophila plants are widely distributed throughout India, and occupy a significant position in traditional systems of medicine. L. indica (Linn.) Druce [7,8] is an aquatic, or nearly aquatic, perennial herb found as submersed, emergent, and amphibious stem plant. The submerged stems are smooth and have leaves up to 30 mm long, feathery, and in whorls about the stem. These differ from the emergent stems, which are covered with flat shiny hairs and have leaves, generally lance-shaped, up to 3 cm long with toothed margins. Stems may be up to 12 feet long. The emergent stems are usually 2–15 cm above the surface of the water. Single white, pink, purple or blue to lavender flowers, sometimes with conspicuous

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spots, occasionally (March–November) occur on the emerged portion of the stem. Sepals have five, green, hairy lobes, each 4–5 mm long. The upper portion is purple and composed of five fused petals forming a tube with two lips — adaxial lip (dorsal) is 2-lobed, while abaxial lip (ventral) is 3-lobed [7–9]. L. indica is widely used in the Indian traditional system of medicine in the treatment of various diseases like pestilent fever, dysentery, dyspepsia, and elephantiasis [9–11]. The plant is used by the local tribal people as carminative and antiseptic. The whole plant is also found to be herbivorous, and hence supposed to be safe or less toxic in animals. L. indica extract was found to inhibit the growth of Xanthomonas campestris and Xanthomonas malvacearum in vitro [12]. Mishra et al. [13] also studied the antimicrobial activity of the same plant extract against a number of bacterial strains including Bacillus, Pseudomonas, Salmonella, Staphylococcus and Xanthomonas species. L. indica has appeared to be a rich source of flavonoids. Previous phytochemical investigations on L. indica resulted the isolation of flavonoid constituents such as 5-hydroxy6,8-di-methoxy-3′,4′-methylene-dioxyflavone [14], 3′,4′ethylenedioxy-5-hydroxy-3-(1-hydroxy-1-methyl ethyl)6,7-dimethyl-5′-methoxyflavone-8-carboxylic acid [15], 5,8dihydroxy-6,7,4′-trimethoxy-flavone [16], 5,2′-dihydroxy-8,3′, 4′-trimethoxyflavone [17], 5,6-dihydroxy-7,8,4′-trimethoxyflavone [17,18], (2S)-5,7,3′,4′-tetramethoxyflavanone [19] and 5,7,2′,5′-tetramethoxy flavones [19] from the whole plants of L. indica so far. Recently, Sandhya et al. [20] carried out GCMS studies of the hydro-distillate of L. indica volatile oil and reported the presence of monoterpenes, essential oils, and long chain fatty acids. As a part of our ongoing studies aimed to phytochemically and pharmacologically characterize the title plant, we isolated for the first time a new pentacyclic triterpene, 3-oxo-olean12(13),18(19)-dien-29α-carboxylic acid (1, Fig. 1) from ethyl acetate extract of aerial parts and roots of L. indica, and evaluated its antimicrobial potential. The structure of 1 was elucidated on the basis of detailed spectral studies including IR, 1H NMR, 13 C NMR, DEPT-135, 2D NMR and MS. The triterpenoid 1 was found to exhibit pronounced antibacterial activity which may be correlated, at least by part, to the established antimicrobial property of the plant. Microbial infectious agents create huge health-hazards to populations, where they cause high morbidity 29

30

COOH 20

19 12

18

22

11 25

26

13

1

17

28

10 3

27

O H 24

23

Fig. 1. 3-Oxo-olean-12(13),18(19)-dien-29α-carboxylic acid (1).

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and mortality [21–24]. Search for novel antimicrobials is needed to overcome the drawbacks of current antimicrobial drugs as well as to obtain more potent agents. In recent years, natural chemotypes have attained renewed interest due to their commendable importance and applications in drug discovery processes [25]. 2. Materials and methods 2.1. General experimental procedures Column chromatography (CC): silica gel (SiO 2; 60– 120 mesh; Merck Chemicals); oleanolic acid (OA, SigmaAldrich); m.p.: Veego melting point apparatus; uncorrected; UV Spectra: Shimadzu UV-3101PC spectrophotometer; IR Spectra: Shimadzu FT-IR 8400S spectrometer; ν in cm−1; 1H and 13C NMR Spectra: Bruker DRX500 NMR spectrometer, at 500 and 125 MHz, resp., in CDCl3, δ in ppm rel. to Me4Si, J in Hz; HRMS (TOF-MS): QTOF Micro mass spectrometer; EI-MS: JEOL-JMS 600 (70 eV) mass spectrometer; in m/z (rel. %); elemental analyses: Elementar Vario EL III Carlo Erba 1108 micro-analyzer instrument; optical rotation: Polarimeter manufactured by Bellingham Stanley Ltd. (Model ADP410). 2.2. Plant materials Whole plants (aerial parts and roots) of L. indica (Linn.) Druce (Scrophulariaceae) were collected in November, 2009 at and around Santiniketan, West Bengal, India, and identified by Dr. H. R. Chowdhury (Botany Department, VisvaBharati University). A voucher specimen (V/LC/LI/5/2009) is preserved in the Laboratory of Natural Products and Organic Synthesis of this University. 2.3. Extraction and isolation Air-dried and defatted whole plants of L. indica (5 kg) were extracted with ethyl acetate in a Soxhlet apparatus for about 70 h; the ethyl acetate extract (~ 4.5 l) was then concentrated in a rotary evaporator, and the concentrated gammy dark-green mass (119 g) was successively fractioned with varying solvents. The reduced mass (23 g) obtained from the petrol ether (60–800) soluble portion was then subjected to column chromatographic (CC) resolution using silica gel (60–120 mesh, 400 g). The petrol-benzene (1:3) eluent afforded the compound 1 as yellowish-white amorphous solid (Yield: 83 mg). 3-Oxo-olean-12(13),18(19)-dien-29α-carboxylic acid (1), yellowish-white amorphous solid, C30H44O3, [M]+ at m/z 452, [α]20 D + 120° (c 1, CHCl3), responded positively to the Liebermann–Burchardt and Zimmermann test; IR, 1H NMR (500 MHz, CDCl3), 13C NMR (125 MHz, CDCl3), DEPT-135, HMQC data are described in the text (also in Table 1). EIMS (70 eV): m/z (% rel) 452 [M]+ (21.14), 407 [M\COOH]+ (32.55), 392 [M\COOH\CH3]+(23.13), 206 (23.13), 246 (100), 231 [246\Me]+ (62.24), 201 [246\COOH]+(20.50), 202 [201 + H]+(28.84), 189 [206\Me\2H]+ (35.40), 184 [231\HCOOH\H]+ (47.96); HRMS(TOF-MS): m/z 475.3190 (C30H44O3Na, [M + Na]+ requires 475.3188); anal. calcd. for C30H44O3: C, 79.60; H, 9.80. Found C, 79.61; H, 9.79.

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Table 1 1 H NMR,

13

C NMR, DEPT-135 and HMQC spectral data of compound 1.

Position

1 13 H-NMR C-NMR (CDCl3, 125 MHz) (CDCl3, 500 MHz) δC-Value DEPT-135 1H–13C-correlation δH-value (HMQC)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

1.06 2.38 – – 1.32 + + – + – 1.97 5.26 – – 1.05 1.26 – – 5.29 – + + 1.12 1.06 1.01 0.83 1.19 0.91 – 1.03

(2H, m) (2H, m)

(1H, m)

(2H, m) (t)

(m) (m)

(s)

(s) (s) (s) (s) (s) (s) (s)

39.62 34.45 218.16 46.88 55.56 19.92 32.79 40.63 50.51 37.13 23.92 125.85 144.02 42.39 26.94 30.06 39.83 138.44 122.66 47.72 30.98 37.04 26.82 21.55 15.37 17.36 26.18 33.45 183.97 21.79

CH2 CH2 C C CH CH2 CH2 C CH C CH2 CH C C CH2 CH2 C C CH C CH2 CH2 CH3 CH3 CH3 CH3 CH3 CH3 COOH CH3

1.06 (H-1) 2.38 (H-2) No coupling No coupling 1.32 (H-5) – – No coupling – No coupling 1.97 (H-11) 5.26 (H-12) No coupling No coupling 1.05 (H-15) 1.26 (H-16) No coupling No coupling 5.29 (H-19) No coupling – – 1.12 (H-23) 1.06 (H-24) 1.01 (H-25) 0.83 (H-26) 1.19 (H-27) 0.91 (H-28) No coupling 1.03 (H-30)

+ are not discernable.

2.4. Microorganisms The bacterial strains used in this study were procured from Microbial Type Culture Collection (MTCC), Institute of Microbial Technology, Chandigarh, India. The bacterial strains used belonged to both Gram-positive and Gram-negative categories. Bacillus subtilis MTCC121 an endospore former, Staphylococcus aureus MTCC96, Listeria monocytogenes MTCC657 were Gram-positive bacteria and Salmonella typhimurium MTCC98, Escherichia coli MTCC1667, Pseudomonas aeruginosa

MTCC741, Pantoea ananatis MTCC2307 were Gram-negative bacteria. One probiotic bacterium Lactococcus lactis subsp. lactis LABW4, isolated in our laboratory, was used to check any negative role of the compound. 2.5. Antimicrobial spectrum The minimum inhibitory concentration (MIC) values of the compound 1 against the different bacteria used (Table 2) were determined from the results of both broth microdilution [26] and counting of colony forming units (CFU) experiments [27]. The broth micro-dilution experiment was performed according to the Clinical and Laboratory Standards Institute (CLSI) guidelines for aerobically grown bacteria in Mueller–Hinton medium [26]. The counting of colony forming units was done by treating the bacteria at different concentrations, plating them after serial dilution and incubating for 24 h at their ideal growth temperatures. A 2% inoculum of early stationary phase culture of each bacterium was used to initiate bacterial growth; oxytetracycline and oleanolic acid (OA; a naturally occurring pentacyclic triterpene having similar skeleton to 1) were used as positive controls for comparison in the experiments. The killing ability of the test compound 1 at its different concentrations was also compared by agar disk diffusion method [28]. Inhibition zone diameters against different bacteria were measured at their respective MIC concentrations as determined earlier. Known amounts of test samples (0–1000 μg per disk) were applied to cellulose paper disk which were later put on the surface of prespreaded lawns of test bacteria and were incubated at 37 °C (S. aureus, P. aeruginosa, B. subtilis, L. monocytogenes, S. typhimurium) or at 28 °C (P. ananatis) for 24 h. To know the effect of the compound 1 on the rate of growth, one representative Gram-positive (B. subtilis) and one representative Gram-negative (P. aeruginosa) bacteria were taken and growth of the bacteria was monitored by measuring the turbidity at 660 nm by treating or non-treating with the compound (Fig. 2: Supplementary data). To determine the mode of action, the test compound was added on actively growing cultures of the same Gram-positive and Gramnegative bacteria followed by counting the colony forming units at different time intervals and plotting the values in graphs (Fig. 3: Supplementary data).

Table 2 Antibacterial activity of compound 1 and the positive controls oxytetracycline and oleanolic acid against the bacteria E. coli, S. typhimurium, S. aureus, B. subtilis, P. aeruginosa, P. ananatis and L. monocytogenes assessed by the disk diffusion and the broth microdilution methods. Values are arithmetic means with ranges in parentheses (n = 3). Microorganisms

Incubation period [h]

Inhibition zone diameter [mm]a

E. coli S. typhimurium S. aureus B. subtilis P. aeruginosa P. ananatis L. monocytogenes L. lactis subsp. lactis LABW4

24 24 24 24 24 24 24 24

22 15 25 28 25 15 25 –b

1

a b c

(20–24) (14–17) (23–27) (26–29) (23–26) (14–17) (24–27)

Minimum inhibitory concentration (MIC) [μg/ml]

Oxytetracycline

Oleanolic acid

1

Oxytetracycline

Oleanolic acid

25 (23–27) 28 (27–29) 21 (19–22) 19 (18–20) 31 (29–32) –b 19 (20–24) 28 (27–29)

19 16 23 21 20 15 21 16

40 100 30 25 30 75 30 –b

10 10 20 20 10 Rc 20 12

50 100 35 30 40 60 30 30

(17–20) (14–17) (22–24) (20–23) (18–21) (14–16) (20–22) (15–17)

Inhibition zone diameters were assessed at corresponding MIC concentrations obtained by microdilution method (200–500 mg/disk). –: No microbial growth inhibition. Resistance.

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2.6. Anti-probiotic bacteria test To check any negative role of the compound 1, it was tested against a probiotic lactic acid bacterium L. lactis subsp. lactis LABW4 by similar agar well diffusion assay as well as by CFU count method as described above. Oleanolic acid used as a natural reference standard was also tested for its activity against the organism. 2.7. Mammalian toxicity test To check any kind of toxicity of the compound 1 on mammalian system, the compound at 200 μg/ml was administered through food to Swiss albino mice with a sample size n = 4 for a week. Blood was collected through cardiac puncture where Triplex III was used as an anticoagulant during drawing of blood. Immediate blood samples were subjected to serum glutamic pyruvic transaminase (SGPT) and serum glutamic-oxaloacetic transaminase (SGOT) assays for liver functions analyses. The tests were performed following standard laboratory methods [29,30]. 2.8. Effect of compound on cellular activity The same representative bacteria of Gram-positive and Gram-negative category were grown till their early stationary phase. They were centrifuged and washed with sterile phosphate buffer. One set of aliquot of each bacterium was treated with compounds at their MIC concentrations. Samples were withdrawn at different hour intervals, centrifuged and the presence of nucleic acid was determined from the supernatant following standard method [31]. 2.9. Scanning electron microscopy (SEM) analysis Bacterial strains treated with compound 1 were collected by centrifugation followed by washing and were scanned for their surface morphology. The specimens were prepared as follows: Cells were harvested by centrifugation at 10,000 ×g for 10 min. Bacterial pellets were washed thrice with normal saline and prefixed with a mixture of 3% glutaraldehyde and 5% DMSO in 0.05 M acetate buffer, pH 5.0, for 30 min, cells were then harvested by centrifugation at 10,000 ×g for 10 min and the pellets were washed thrice with 0.1 M sodium acetate buffer, pH 5.0. The pellets were then post-fixed with osmium tetra oxide solution for 30 min. Cells were collected by centrifugation at 10,000 ×g for 10 min and were dehydrated with a series of ethanol starting with 30% via 40, 50, 60, 70, 80, 85, 90, 95% and finally with absolute ethanol with 10 min of dehydration in each grade. The cells were then spread on a clear glass slide (1 cm2). The slide was mounted on a stub with double side adhesive tape and silver dag and coated slowly with a very thin (2–5 nm) layer of gold in a sputtering unit prior to examination under scanning electron microscope (Philips, PSEM-500, Holland) following the method of Sarem-Damerdji et al. [32]. 3. Results and discussion Compound 1 had a molecular formula C30H44O3 as deduced from its elemental analyses as well as from HR-TOF-MS

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([M + Na]+, 475.3190); the compound 1 exhibited the molecular ion peak [M]+ at m/z 452 in its EIMS spectrum that is also consistent with the molecular formula C30H44O3. It responded positively to the Liebermann–Burchardt test for pentacyclic triterpene. The IR spectrum of 1 showed characteristic absorption bands at 3380–3490 (\OH of carboxylic functionality), 1697 (carbonyl of both oxo and carboxylic functionality), 1458 and 964 (tri-substituted double bond) cm− 1. The 1H NMR spectrum displayed signals for (i) seven tertiary methyls at δ 0.83, 0.91, 1.01, 1.03, 1.06, 1.12 and 1.19 (3H each, s, 7 × CH3); (ii) a two proton multiplets at δ 2.38 assignable to keto-methylene protons; (iii) two vinylic protons at δ 5.26 (1H, t) and δ 5.29 (1H, s) associated with two tri-substituted double bonds. The remaining protons appeared as complex multiplets between δ 1.2 to 1.8. The olean-type skeleton and the presence of these functionalities in the triterpenoid molecule received excellent support from the appearance of the chemical shifts in its 13C NMR spectrum that recorded carbon signals for thirty different carbons; the 13C-chemical shift values and their respective DEPT-135 analysis clearly indicated an olean-type of skeleton for the compound 1 having seven methyls, nine methylenes, two methines, and six quaternary carbons (Table 1). The functionalities present in 1 received full supports from its 13C NMR spectral analysis: δ 218.16 (oxo), 183.97 (COOH), 125.85 & 144.02 and 122.66 & 138.44 (for two separately distinct tri-substituted NC_Cb bonds). The EIMS fragmentation pattern of the triterpenoid 1 (Scheme 1: Supplementary data) is very similar to that of Δ12-oleanaene type of pentacyclic triterpenoids [33]. The significant mass fragments at m/z 206 & 189 clearly suggest that the oxo group is in the A/B ring portion, while the mass peaks at m/z 246 (base peak), 231, 201 & 184 indicate the location of carboxyl function and another C_C bond in the portion containing D/E rings of the triterpene nucleus. The appearance of significant mass ion peaks at m/z 392 ([M\Me\COOH]+, 23.13%), 246 (retro-Diels-Alder fragmented mass-ion peak, 100%), 231 ([246\Me]+, 62.24%), 201([246\ COOH]+, 20.56%), 184 ([231\COOH]+, 47.96%) are indicative of the presence of Δ12(13),18(19)-oleanadiene type of pentacyclic triterpene having a methyl at C(28) and a carboxylic (COOH) group at C(20) position (Scheme 1: Supplementary data). On the other hand, biogenetic precedence as well as positive Zimmermann test conclusively locates the oxo-functionality in the A/B ring portion of the triterpene molecule at C(3). The overall spectral properties along with careful scrutiny of the 13C NMR data (Table 1) for 1, comparable to the values as reported for the compounds having similar skeletons [34–38], thus led the present investigator to formulate the triterpenoid compound unequivocally as 3-oxo-olean12(13),18(19)-dien-29α-carboxylic acid (1). This contention was received further verification from its 2D NMR correlation spectroscopy; the HMQC spectrum of 1 showed the expected 1 H–13C correlations (Table 1). The antibacterial effect of the compound 1 was evaluated against three Gram-positive (B. subtilis MTCC121, S. aureus MTCC96, L. monocytogenes MTCC657), L. lactis subsp. lactis LABW4 and four Gram-negative (S. typhimurium MTCC98, E. coli MTCC1667, P. aeruginosa MTCC741, P. ananatis MTCC2307) bacteria. The compound was found to kill all the seven bacteria at different concentrations. The minimum inhibitory

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concentrations (MICs) of the compound against different bacteria were determined primarily based on the results of microdilution method (Table 2) and by measuring the colony forming units (Table 3: Supplementary data). These results were later also compared by agar disk diffusion method (Table 2). Oxytetracycline and oleanolic acid were used as positive controls to compare the efficacy of the compound. Compound 1 showed its activity against Gram-positive bacteria at very low concentrations within a range of 25–30 μg/ml, whereas it showed a variable sensitivity against Gram-negative bacteria. It could kill E. coli and P. aeruginosa at lower concentrations (40 and 30 μg/ml, respectively), but a comparatively higher amounts (75 and 100 μg/ml, respectively) of the compound were required to kill P. ananatis and S. typhimurium. The results were expressed at average of at least three observations with comparatively standard errors given in parenthesis in Table 2. The results of Table 3 (Supplementary data) offer an idea about the count of viable organisms at different concentrations of the compound. Abrupt reduction in CFU counts was observed for each organism at their respective MIC values. Oxytetracycline, a broad-spectrum antibiotic, was used as a reference positive control that showed much lower values of MIC for each bacterium. Beyond oxytetracycline, we also screened oleanolic acid (a naturally occurring and biologically potent pentacyclic triterpene having similar skeleton to 1) to study a comparative antibacterial potential. To our delight, compound 1 exhibited comparable antibacterial potential with that of the well-regarded natural molecule with relatively lower MIC values against E. coli, B. subtilis and P. aeruginosa (Table 2). A good number of natural triterpenoids are reported in literature to possess a wide range of antimicrobial activities; however, in most of the cases to get effective results a comparatively higher amount of compounds are usually required. Scalon Cunha et al. [39] evaluated some natural triterpenoids including oleanolic and ursulic acids including some of their derivatives as well with MIC values in the range of 30 to 80 μg/ml against different Streptococcus species. Prachayasittikul et al. [40] also studied antimicrobial activity of certain bioactive triterpenoids against Gram-positive pathogens (MICs 64–256 μg/ml). Recently, Kipilano et al. [41] isolated several triterpenoids from Veronica auriculifera, which could kill both Gram-positive and Gram-negative pathogenic bacteria with MIC values around 250 μg/ml. All these observations attest the similar spectrum of antimicrobial activities by the triterpenoids. In our present study it is noteworthy that unlike compound 1, oleanolic acid inhibited the growth of probiotic lactic acid bacterium L. lactis subsp. lactis LABW4 with MIC value of 30 μg/ml. The bacterial strains used in the present study represent human pathogenic, food spoilage and plant pathogenic groups that cause serious damage to mankind. Considering low MIC values of the present compound against the organisms studied, it could be considered as a prospective candidate in the on-going antibacterial drug-discovery programs, and also as an effective food preservative. The ability of the compound to kill L. monocytogenes, a potential food spoilage organism further endorsed this possibility. Mode of action of the compound was also assessed by applying its MIC to the actively growing culture followed by CFU count in the following hours. The results are presented in Fig. 3 (Supplementary data) that

shows sharp decline in the CFU count within a few hours. The results clearly indicate its bactericidal mode of action against both B. subtilis and P. aeruginosa. The effect of compound 1 on the rate of bacterial growth was monitored against two bacteria (B. subtilis and P. aeruginosa). The test compound slows down the growth rate effectively as determined by measuring optical density of the culture broth in the presence and absence of 1; for both of the bacterial strains, optical density was found to increase very slowly in the presence of the test compound in comparison to their respective controls (Fig. 2: Supplementary data). A similar mode of action by a 6-oxophenolic triterpenoid with sharp decline in CFU count upon treatment on exponentially grown B. subtilis cells was observed; the investigators pointed out the probable action of the compound on bacterial cell wall and membrane integrity and biosynthesis [42]. The triterpenoid compound 1 under present study also points its activity in the same line. Fig. 4 (Supplementary data) indicates the effect of the test compound 1 on cellular activity. Many compounds because of their ability to bind with membrane components of bacteria disrupt the integrity of membrane structure [43] which leads to loss of vital metabolites from cell and becomes responsible for death of the cells. Loss of nucleic acid is one of such activity which we have found in two of the treated bacteria by the compound (Fig. 4: Supplementary data). As nucleic acids are the main information transferring molecules of any cell, thus its loss would definitely affect the cell viability. Loss of other molecules such as lactate dehydrogenases by exogenous treatment of compounds are also reported [31] indicating the involvement of membranes due to compound treatment. Scanning electron micrographs (SEMs) clearly indicate the effect of the compound 1 on different bacterial cells upon treatment. Morphological variations and especially the wrinkling on cell surface is very prominent feature observed for eight or 24 h of treatment with compound 1 on E. coli, P. aeruginosa, B. subtilis and L. monocytogenes (Fig. 5). Arrangement of cells in different patterns viz., end to end forming pseudo-thread like structures, sidewise attachments are also observed (Fig. 5). Change of morphology of bacteria by triterpenoid treatment was also observed earlier [42]. No remarkable change in CFU count of the lactic acid bacterium L. lactis subsp. lactis LABW4 was observed even at 500 μg/ml of the test compound which was 9.1 × 107 from an initial count of 4.2 × 108. The inability of the compound to kill or reduce the viable bacterial count of the probiotic lactic acid bacterium by agar well diffusion and CFU count method indicates its applicability in a prospective way (Table 2, and Table 3 as Supplementary data). To verify whether the compound can exert any toxic effect to mammalian system, it was administered more than its antibacterial MIC concentrations to Swiss albino mice model along with their food for a week. Blood was drawn from these treated and untreated animals and two sensitive indicator enzymes viz., serum glutamic oxaloacetic transaminase (SGOT) and serum glutamic pyruvic transaminase (SGPT), for liver function was assayed from the blood samples. Most of the toxic compounds when entered in the mammalian system are either metabolized to its non-toxic forms in liver or are excreted out. In many occasions, liver cannot metabolize the toxic molecules and the molecules in turn damage liver-cells significantly. As a result, SGOT and SGPT are released in higher quantities in blood. The levels of

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A

Untreated control cells

Cells treated for 8 hrs

Cells treated for 24 hrs

B

Untreated control cells

Cells treated for 8 hrs

Cells treated f or 24 hrs

Untreated control cells

Cells treated for 8 hrs

Cells treated for 24 hrs

Untreated control cells

Cells treated for 8 hrs

Cells trea ted for 24 hrs

C

D

Fig. 5. Effect of compound 1 on the morphology of A) Escherichia coli, B) Pseudomonas aeruginosa, C) Bacillus subtilis and D) Listeria monocytogenes assessed by scanning electron microscopy.

these two enzymes in the treated mice blood were not changed in comparison to untreated samples. This indicates the non-toxic nature of the compound on mammalian system as usually observed for nontoxic compound [44]. 4. Conclusions A new pentacyclic triterpenoid, characterized as 3-oxoolean-12(13),18(19)-dien-29α-carboxylic acid (1) on the basis of detailed spectral studies, was isolated from the aerial parts and roots of L. indica (Scrophulariaceae), and the isolated compound was evaluated to possess considerable antibacterial activity against a wide range of bacterial strains including three Gram-positive, four Gram-negative, one plant pathogenic

and one human pathogenic bacteria. To our delight, the new triterpene 1 also exhibited comparable antibacterial potential with that of oleanolic acid with relatively lower MIC values against E. coli, B. subtilis and P. aeruginosa. Compound 1 inhibited the growth of Gram-positive B. subtilis and Gram-negative P. aeruginosa completely with a clear bactericidal mode of action at their respective MIC values of 25 and 30 μg/ml. The compound upon treatment on both B. subtilis and P. aeruginosa released substantial amount of nucleic acid in the external medium and also effected the change of morphology towards pleomorphicity as shown from their SEM experiments, thereby indicating its probable action on cell membrane. Furthermore, unlike oleanolic acid the triterpenoid 1 was found not to inhibit a probiotic lactic acid bacterium L. lactis subsp. lactis

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LABW4 under in vitro conditions, and also to possess no toxicity in Swiss albino mice. The bacterial strains used in the present study represent human pathogenic, food spoilage and plant pathogenic groups. They cause serious damage to mankind by their activities. Considering low MIC values of the present compound against the organisms studied, it could be considered as a prospective candidate in the on-going antibacterial drug-discovery programs, and also as an effective food preservative. The ability of the compound to kill L. monocytogenes, a potential food spoilage organism further endorsed this possibility. Oleanolic acid is a promising natural molecule and is anticipated to play important role in the domain of ongoing drug research. Besides potent antimicrobial activities, oleanolic acid and its semisynthetic derivatives are reported to possess a wide range of pharmaceutical activities such as cytotoxic, anticancer, antiproliferative, antioxidant, anti-inflammatory, anti-HIV, antidiabetic, antipruritic, antiallergic and many more [45–58]; hence, the present study offers a new chemical entity with comparable antibacterial potential with that of oleanolic acid with a hope that this report would draw the attention of the scientific community at large for further in-depth research to explore possible pharmaceutical applications of the molecule itself and its semi-synthetic analogs. Conflict of interest There is no conflict of interest to declare. Acknowledgments The authors are thankful to the Bose Institute, Kolkata for spectral measurements. They are also grateful to the UGC, New Delhi for financial support. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.fitote.2013.07.012. References [1] Li HL. Flora of Taiwan. In: Li HL, Liu TS, Huang TC, Koyama T, Devol CE, editors, vol. IV. Taiwan: Epoch Publishing Co., Ltd.; 1978, p. 551–616. [2] Matsumura J, Hayata BJ. Enumeratio Plantarum Formosanarum. J Coll Sci Imp Univ Tokyo Jpn 1906;22:277. [3] Philcox D. A taxonomic revision of the genus Limnophila R.Br. (Scrophulariaceae). Kew Bull 1970;24:101–70. [4] Yamazaki T. A revision of the genera Limnophila and Torenia from Indochina. J Fac Sci Univ Tokyo III 1985;13:575–624. [5] Yang YP. A synopsis of aquatic angiospermous plants of Taiwan. Bot Bull Acad Sin 1987;28:191–209. [6] Brahmachari G. Limnophila (Scrophulariaceae): chemical and pharmaceutical aspects. Open Nat Prod J 2008;1:34–43. [7] Chopra RN, Nayer SL, Chopra IC. Glossary of Indian medicinal plants. New Delhi: CSIR; 1986, p. 154. [8] Sivarajan VV, Balachandran I. Botanical notes on the identity of certain herbs used in Ayurvedic medicines in Kerala. III. Hribera and amragandha. Anc Sci Life 1986;5:250–4. [9] Ambasta SP. The useful plants of India. New Delhi: PID, CSIR; 1986, p. 329–335. [10] Satyavati GV, Gupta AK, Tandon N. Medicinal plants of India, vol. 2. New Delhi: Cambridge Printing Works, ICMR; 1987, p. 166. [11] Thammanna, Narayana RK, Madhava CK. Angiospermic wealth of Tirumala. Tirupati: TTD Press; 1994, p. 115.

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