Songklanakarin J. Sci. Technol. 37 (1), 43-48, Jan. - Feb. 2015

http://www.sjst.psu.ac.th

Original Article

In vitro evaluation of the antibacterial and anti-inflammation activities of lansium (Lour.) Skeels

Pirasut Rodanant1*, Rudee Surarit2, Surat Laphookhieo3, and Jintakorn Kuvatanasuchati4

1 Department of Advanced General Dentistry,

2 Department of Oral Biology, Faculty of Dentistry, Mahidol University, Ratchathewi, Bangkok, 10400 Thailand.

3 Natural Products Research Laboratory, School of Science, Mae Fah Luang University, Mueang, Chiang Rai, 57100 Thailand.

4 Department of Microbiology, Faculty of Dentistry, Chulalongkorn University, Pathum Wan, Bangkok, 10330 Thailand.

Received: 25 July 2014; Accepted: 17 November 2014

Abstract

Crude extracts of twigs and roots of Clausena lansium (Lour.) Skeels were separated and purified using repeated silica gel column chromatography to yield 12 compounds identified as xanthotoxol (1), imperatorin (2), heraclenol (3), heraclenin (4), wampetin (5), indicolactonediol (6), murrayanine (7), O-demethylmurrayanine (8), indizoline (9), 3-formyl-6-methoxycarbazole (10), lansine (11) and glycozolidal (12). All pure compounds were tested for their antibacterial and anti-inflammation activities using disc diffusion method and enzyme-linked immunosorbent assay (ELISA), respectively. At a concentration of 50 µg/mL, three carbazole alkaloid components (compounds 10, 11, and 12) demonstrated moderate antibacterial activity against the periodontopathic bacteria, Porphyromonas gingivalis. Compound 10 and the crude extract of twig revealed impressive anti- inflammation potency. From these results, selected carbazole alkaloid compounds from Clausena lansium might be potential raw products in generating new anti-inflammation and antibacterial agent used as an adjunctive medication in treating periodontal disease.

Keywords: Clausena lansium, carbazole alkaloid, antibacterial, anti-inflammation, periodontal disease

1. Introduction thousands of diverse chemical compounds with different bio- logical activities (Balandrin et al.,1985). This might be strong The emergence of antibiotic resistance and undesir- evidence supporting the successful treatment of various able effects due to the use of synthetic chemicals stress the diseases using edible (Raskin et al.,2002). Periodontal importance of finding alternative agents to treat various disease, a chronic inflammatory disease, is considered a diseases. In vitro phytochemical studies of higher plants major oral problem since fifteen percent of the global popula- reveal that these plants are the source of hundreds of tion is suffering from its severe form. The accumulation of various bacterial species, bacterial toxins, and inflammatory cytokines in the site of periodontal inflammation, accounting for the disease initiation, render the importance of the usage * Corresponding author. of antibacterial and anti-inflammation drugs to slow and/or Email address: [email protected] halt the disease progression. Clausena lansium (Lour.) 44 P. Rodanant et al. / Songklanakarin J. Sci. Technol. 37 (1), 43-48, 2015

Skeels known in Thai as “Mafai jean” is an indigenous described previously (Maneerat et al., 2010a; 2010b; 2012) in the Southeast region. It is a slow growing, strongly (Figure 1). Each of the isolated compounds and crude extract scented evergreen plant belonging to the family from twig were dissolved in dimethyl sulfoxide (DMSO, and can reach 3 to 8 meters height. Previous studies of its Sigma, St. Louis, U.S.A.) to obtain the solutions of 1 mg/mL bioactivities showed that compounds isolated from its parts (w/v) concentration. The stock solutions were kept at -20°C. had antioxidant, anti-inflammation, antifungal, hepatoprotec- tive, and anticancer activities (Adebajo et al.,2009; Ng et 2.3 Cell line and Chemicals al.,2003; Prasad et al.,2009). Therefore, it is of interest to investigate the antibacterial and anti-inflammation activities The monocyte cell line U937 was kindly provided by of the crude extract from twig and isolated compounds from the Siriraj Medical School, Thailand. Human gingival fibro- twig and root of Clausena lansium (Lour.) Skeels. blast (HGF) cell line (ATCC CRL-2014) was purchased from ATCC (U.S.A.). Growth media (RPMI 1640 containing 2 mM 2. Materials and Methods L-glutamine and Dulbecco’s Modified Eagle’s Medium (DMEM)) and antibiotic-antimycotic solution were purchased 2.1 Plant materials from Gibco (Grand Island, NY, U.S.A.). Fetal bovine serum (FBS), Trypan blue, 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl- Twigs and roots of Clausena lansium (Lour.) Skeels tetrazolium bromide (MTT), cell culture grade dimethyl- were collected from Muang District, Nan Province, Thailand. sulphoxide (DMSO), LPS (Esterichia coli 026:B6), and The identification of the plant was performed by one of the phorbol-12-myristate-13-acetate (PMA) were purchased from author (S.L.) through comparison with a voucher specimen Sigma-Aldrich Co. (St. Louise, MO, U.S.A.). Dexamethasone no. QBG 25077 in the herbarium collection of Queen Sirikit (DM) was purchased from APP Pharmaceuticals, LLC Botanic Garden, Mae Rim, Chiang Mai, Thailand. (Schaumberg, IL, U.S.A.). ELISA kit was purchased from Thermo Scientific (Rockford, IL, U.S.A.). 2.2 Extraction and Isolation 2.4 Cell culture All pure compounds, including xanthotoxol(1), imperatorin (2), heraclenol (3), heraclenin (4), wampetin (5), The HGF cells and U937 cells were maintained in indicolactonediol (6), murrayanine (7), O-demethylmurray- DMEM and RPMI 1640, respectively. Each medium was anine (8), indizoline (9), 3-formyl-6-methoxycarbazole (10), supplemented with 100 U/mL of penicillin, 100 g/mL of lansine (11) and glycozolidal (12), were isolated from the twigs streptomycin and 10% fetal bovine serum. Cells were grown

(compound 1-7) and roots (compound 8-12) of C. lansium as at 37°C and 5% CO2 in humidified air.

Figure 1. The chemical structures of isolated compounds P. Rodanant et al. / Songklanakarin J. Sci. Technol. 37 (1), 43-48, 2015 45

2.5 Cytotoxic activity 2.8 Anti-inflammation activity

Stock solutions of plant extracts were diluted on the 2.8.1 Control establishment day of the experiment using appropriate culture medium to the concentration ranging from 10 - 100 µg/mL. The final There were three kinds of control groups as follows: concentration of DMSO in each sample did not exceed 1% positive control consisting of 0.5 µg/mL dexamethasone v/v (Prayong et al., 2008). The cytotoxic activity of the (DM) and 0.5 µg/mL lipopolysaccharides (LPS) from E. coli; extracts were tested with HGF and U937 cells using the cell stimulated by LPS from E. coli (0.5 µg/mL) without any MTT method (Mosman, 1983) with minor modifications intervention was used as blank; and cell incubated by (Kuvatanasuchati et al., 2011). DMEM or RPMI1640 medium was regarded as normal group. Briefly, the cell were seeded in 96 well-plates (200 µl/well at a density of 1×105 cells/ml) incubated at 37°C in 5% 2.8.2 Cellular model establishment and intervention

CO2 atmosphere for 24 h. At 24 h, cells were washed with sterile phosphate buffer (PBS) 100µl and treated with 100 µl Before LPS treatment, the cells (400 µL/well at con- of plant extract solution at various concentrations (five wells centration of 1x105 cell/mL) were inoculated into 24 micro- for each treatment) then incubated at 37°C in 5% CO2 well plates. Then later, after the cells had adhered to the atmosphere for another 24 h. The plate was washed twice bottom of the well (24 hours for HGF and 48 hours for U937), with sterile PBS100 µl of MTT solution (0.5 mg/ml) added to the cell supernatants were disposed and prepared non- each well and incubated in the dark at 37°C for another cytotoxic concentrations of plant fractions were added (400 2 hours. The formazan crystals were solubilized with 100 µl µL/well). Two hours later, 1 µg/mL of LPS from E. coli (400 of DMSO and left for 30 minutes at room temperature. The µL/well) was added into each well. After stimulation for 24 h, absorbance was read at 540 nm by a microplate reader (Model the supernatants were harvested and were kept at -80°C until series UV 900 Hdi, U.S.A.).Three separate experiments were further use. performed. 2.8.3 ELISA assay of TNF- 2.6 Microorganisms The assay consists of four incubations and three The antibacterial activities of all isolated compounds wash cycles. The harvested supernatants were added into were determined against Porphyromonas gingivalis strain designated wells of coated 96-well microplate containing ATCC 33277(Pg ATCC 33277). The medium used for the sample diluents. Plate was covered and incubated for 1 h at activation of the microorganisms was Brain heart infusion room temperature then washed three times with deionized broth (BHI). Brucella (Oxoid®, U.K.) was used as cultivation water. The biotinylated antibody was then added followed by media. The microorganisms were inoculated into BHI and the streptavidin horseradish peroxidase. After each substrate incubated at 37°C in 5% CO2 atmosphere (Anaerobic system; was added, the plate was covered and left for 30 minutes at Forma Scientific, Inc.) for 7 days. The bacterial suspension room temperature then washed. Lastly TMB substrate was was then diluted with BHI broth medium to obtain approxi- added and incubated in the dark for 30 minutes at room mately 1×108cfu/mL. This working concentration obtained temperature. Absorbance was measured by microplate reader transmittance comparable to 0.5 McFarland turbidity standard (Model series UV 900 Hdi, U.S.A.) at 450 and 550 nm. using spectrophotometer at 540 nm. Calculation of the relative absorbance units and the TNF- concentration (pg/ml) for each sample, as well as 2.7 Antibacterial activity construction of the standard curve of recombinant mouse TNF- calibration curves, were performed as described in The growth inhibition tests were performed using the instruction manual. Each sample was measured in dupli- agar diffusion technique. One hundred microlitre (µL) of BHI- cate and concentrations were derived from the standard suspended microorganism was distributed on the agar curve. medium (25 mL/plate) using small-size glass beads. Once the agar surface was dried, paper discs 6 mm in diameter 3. Results (Whatman International, U.K.) soaked with 10 µL of the isolated compound solution (10µg/mL) on each side of the 3.1 Cytotoxic activity disc, were placed on the agar surface. Chlorhexidine 2% and DMSO were used as positive and negative control respec- The cytotoxicity of isolated compounds from tively. Each plate was incubated in anaerobic chamber at Clausena lansium (Lour.) Skeels extract on both cell types

37°C in 5% CO2 atmosphere for 7 days. All tests were was shown in Table 1. The sensitivity of both cells to all performed in triplicate and the antibacterial activity was compounds tested was in the same range. It was found that expressed as the diameters (mean±SE) of inhibition zone the IC50 of all compounds tested with HGF was in the range (mm) produced by the isolated compound. of 29.38 to 128.62 µg/mL and 38.19 to 103.04 µg/mL for 46 P. Rodanant et al. / Songklanakarin J. Sci. Technol. 37 (1), 43-48, 2015

Table 1. In vitro cytotoxicity of isolated compounds from C. alkaloids from C. lansium possessed a moderate growth lansium against human gingival fibroblasts and inhibition activity on a gram-negative periodontopathic monocytes (U937) bacteria, P. gingivalis. The chemical structure of all carbazole alkaloids with antibacterial activity (compounds 10, 11, and

IC50 (µg/mL) 12) was occupied by the formyl group and methoxy group at Compounds the position C-3 and position C-6, respectively. Since the HGF U937 potency of the three active carbazole alkaloids showed differ- 1 106.61±0.29 80.36±0.20 ently, the ring substituents might be responsible for the 2 74.48±0.23 58.77±0.40 bioactivity of the pure compounds. Evidently, additional 3 123.05±0.26 95.76±0.30 aromatic hydroxyl group promoted the antibacterial activity 4 123.28±0.28 96.35±0.16 of some plants (Kim et al., 2012). The same phenomenon was 5 109.42±0.29 83.09±0.01 6 128.62±0.06 103.04±0.67 Table 2. Antibacterial activity of isolated compounds 7 38.36±0.17 38.19±0.07 in paper disc method against Porphyromonas 8 29.38±0.58 39.97±0.06 gingivalis 9 Nil Nil 10 70.20±0.15 56.55±0.16 Compounds Diameters of inhibition zone (mm) 11 79.01±0.22 61.61±0.16 12 120.86±0.38 97.74±0.13 1 0 2 0 3 0 monocytes U937. Only compound 9 showed toxicity effect 4 0 on both cells. 5 0 6 0 3.2 Antibacterial activity 7 0 8 0 Only some carbazole alkaloids (compounds 10, 11, and 10 14±0.62 12) demonstrated antibacterial activity against P. gingivalis 11 20±0.94 but the potency was lower than that of 2% chlorhexidine 12 12±1.16 digluconate (positive control) (Table 2). Compound 11 gave CHX 2% 30±0 the highest antibacterial activity with the diameter of inhibi- DMSO 0 tion zone of 20 mm. Compounds 10 and 12 demonstrated the same growth inhibition efficacy but were less effective than CHX 2% Chlorhexidine digluconate 2% compound 11. Other compounds showed the same result as DMSO Dimethyl sulfoxide negative control (DMSO). Table 3. Effects of isolated compounds from C. lansium on 3.3 Anti-inflammatory activity TNF-á release

After incubation with monocytes followed by LPS Compounds TNF (pg/mL) stimulation, almost all components ineffectively inhibited the release of cytokine except 3-formyl-6-methoxycarbazole 1 1795.5 (compound 10) and the crude extract from twigs. At a con- 2 1847.15 centration of 50 µg/mL, compound 10 showed a TNF 3 1763.73 suppression capacity comparable to the positive control 4 1776.35 (dexamethasone) while the crude extract from twigs demon- 5 1787.03 strated exceptional result. Other compounds seemed to 6 1842.68 increase the expression of this pro-inflammatory cytokines 7 1876.35 (Table 3). 8 1355.93 10 378.15 4. Discussion 11 1444.38 12 1810.23 According to previous phytochemical studies, carba- Crude extract of the twig 17.7 zole alkaloids from plants in the family Rutaceae expressed antimicrobial activity (Arbabet al., 2012; Chakraborty et al., LPS 1098.45 1995; Rahman and Gray, 2005; Wu and Furukawa, 1982). DMSO 144.53 It was demonstrated in our study that several carbazole Dexa 343.78 P. Rodanant et al. / Songklanakarin J. Sci. Technol. 37 (1), 43-48, 2015 47 found in this study since carbazole alkaloids with an addi- tory, hepatoprotective and antioxidant effects. Journal tional hydroxyl group (-OH) on C-2 (compound 11) possessed of Ethnopharmacology. 122, 10-19. the most potent antibacterial activity. Though furanocou- Amiot, F., Fitting, C., Tracey, K.J., Cavaillon, J.M. and Dautry, marins demonstrated antimicrobial activities against various F. 1997. Lipopolysaccharide-induced cytokine cascade strains of gram-negative bacteria (Dongfack et al., 2012), our and lethality in LT/TNF deficient mice. Molecular study did not reveal the same result wherein there was no Medicine. 3, 864-875. such antibacterial activity as determined by the inhibition Arbab, I.A., Abdul, A.B., Aspollah, M., Abdullah, R., zone. Abdelwahab, S.I., Ibrahim, M.Y. and Ali, L.Z. 2012. As tumor necrosis factor- (TNF-) mediates the A review of traditional uses, phytochemical and phar- production of many other cytokines during inflammation macological aspects of selected members of Clausena (Amiot et al., 1997), in this study, the in vitro model of genus (Rutaceae). Journal of Medicinal Plants Re- macrophage-mediated inflammatory event was chosen to search. 6, 5107-5118. assess the effect of crude extract and pure compounds from Balandrin, M.F., Klocke, J.A., Wurtele, E.S. and Bollinger, C. lansium on the production of TNF-. Tested coumarins W.H. 1985. Natural plant chemicals: Sources of indus- and almost all carbazole alkaloids showed no inhibitory trial and medicinal materials. Science. 228, 1154-1160. effects on TNF- production. Nevertheless, compound 10 Chakraborty, A., Saha, C., Podder, G., Chowdhury, B.K. and showed comparable efficacy to dexamethasone and most Bhattacharyya, P. 1995. 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Aromatic hydroxyl explanation for the difference in the potency of the studied group plays a critical role in antibacterial activity of compounds which possessed different side chain on the the curcumin analogues. Natural Product Communi- aromatic ring. cations. 7, 57-58. The antibacterial and anti-inflammation activities of Kumar, K., Pawar, A.P. and Sivakumar, T. 2012. Synthesis and the carbazole alkaloids from C. lansium described in this investigation of anti-microbial and anti-inflammatory study seem to justify the ethnobotanical use of this plant activity of substituted flavones. International Journal which might have a potential for the development of a drug of Research in Pharmaceutical and Biomedical Sciences. for the treatment of inflammation. Due to their anti-perio- 3, 784-791. dontopathogen activity, these compounds may be of interest Kuvatanasuchati, J., Laphookhieo, S. and Rodanant, P. 2011. for use in the treatment of periodontal diseases. 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