Antibacterial activity of two triterpenes from stem bark of Protorhus longifolia

Antibiotic resistance of pathogenic strains has hugely contributed to the wide spread of new and reemerging infectious diseases. There is thus the need for the discovery of new (non-penicillin based) antibiotics. The triterpenes [3β-hydroxylanosta-9,24-dien-21-oic acid (1) and methyl-3β-hydroxylanosta9,24-dienoate (2)] isolated from stem bark of Protorhus longifolia (Benrh.) Engl. were evaluated for their antibacterial activity against a panel of selected general and antibiotic resistant Gram positive and Gram negative bacteria. The antibacterial activity of the compounds was determined using disc diffusion method. The compounds exhibited antibacterial activity against most of the tested bacteria with minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values ranging from 0.16 to 5.00 mg/ml and 0.63 to 5.00 mg/ml, respectively. The triterpenes did not exhibit any bacterial DNA damaging effects, but apparently affected the microbial cell membrane integrity. The triterpenes could be a potentially effective antimicrobial agent to combat infectious diseases.


INTRODUCTION
Pathogenic microorganisms are the major cause of a wide range of infectious diseases, with high rate of mortality in humans resulting from bacterial infections (Tajbakhsh et al., 2011).Despite great progress made in the discovery of antibiotics, the development of bacterial resistance to some of the current antibiotics is a serious global challenge (Hoffmann et al., 2011;Chung et al., 2013).This challenge has then triggered a search for new effective antimicrobial compounds with novel mechanisms of action (Rojas et al., 2003;Zakaria et al., 2009).
Plants have always been an untapped source to provide bioactive compounds as potential therapeutic agents, including antimicrobials.Thus, the wide chemical diversity of plant-derived compounds is important in the development of effective agents to combat infectious diseases resistant to conventional drugs (de León et al., 2005).
Protorhus longifolia (Benrh.)Engl.(Anacardiaceae) is a tall, ever green indigenous tree in Southern Africa.Stem bark of the plant has been traditionally used by the Zulus to cure various diseases such as heartwater and diarrhea in cows (Dold and Cocks, 2001).Antimicrobial (Suleiman et al., 2010) and anti-platelet aggregation (Mosa et al., 2011) activities of the crude extracts of the plant parts have been reported.Lanosteryl triterpenes isolated from the stem bark of P. longifolia have been reported to exhibit anti-platelet aggregation activity (Mosa et al., 2011).
Triterpenes are a versatile group of biologically active plant secondary metabolites widely distributed in the plant kingdom.They are predominantly found on plant surfaces such as leaf, stem bark and fruit waxes (Jäger et al., 2009).These compounds reportedly exhibit a wide spectrum of biological activities including anti-inflammatory (Ko et al., 2007;Mosa et al., 2011), antitumor (Gonzalez et al., 2002) and antimicrobial activity (Motlhanka et al., 2010;Kiplimo et al., 2011).The importance of plant derived triterpenes as new targets for drug development cannot be overemphasised.In this study, we evaluated antimicrobial activity of the triterpenes (3βhydroxylanosta-9,24-dien-21-oic acid, and methyl-3βhydroxylanosta-9,24-dienoate ) isolated from stem bark of P. longifolia.

Reagents
Unless otherwise stated, all chemicals and reagents (of analytical grade) used were obtained from Sigma-Aldrich Chemical Co.(St Louis, MO, USA).

Plant collection
Fresh stem barks of P. longifolia were collected in March, 2012 from KwaHlabisa, KwaZulu-Natal, South Africa.The plant with the voucher specimen RA01UZ was confirmed by Mrs. N.R. Ntuli, Department of Botany, University of Zululand.The plant material was well cleaned with tap water, chopped into smaller pieces and air-dried.It was then powdered (2 mm mesh) and stored in a sterile brown bottle at 4°C till processing.

Extraction and isolation
The methods previously described by Mosa et al. (2011) were followed to extract and isolate the two triterpenes from stem bark of P. longifolia.Briefly, the powdered stem bark was defatted with nhexane and the residue then extracted (1:5 w/v) with chloroform.Silica gel column chromatography (24 × 700 mm; Silica gel 60; 0.063 to 0.2 mm; 70 to 230 mesh ASTM; Merck, Darmstadt, Germany) was used to isolate the compounds from the chloroform extract (13 g).A hexane-ethyl acetate solvent gradient system (9:1 to 3:7) was used as the mobile phase, collecting 20 ml fractions.The collected fractions, analysed by thin layer chromatography (TLC) (silica gel 60 TLC aluminum sheets 20 cm × 20 cm, F254) were combined based on their profile to yield 18 combined fractions (Fr.A-R).The ninth and fourteenth combined fractions were further separately purified with hexane and ethyl acetate to afford compounds 1 (0.72 g) and 2 (1.14 g), respectively.Stuart SMP 11 melting point apparatus (Shalom Instruments supplies, Durban, R.S.A) was used to determine melting point.Structures of the triterpenes were established and confirmed through the use of spectral techniques such as infrared (IR) (KBr, Perkin-Elmer 100 FTIR),1D and 2D nuclear magnetic resonance (NMR) techniques ( 1 H-1 H, 13 C-13 C, DEPT, COSY, HMQC, HMBC and NOESY) (in CDCl3, Bruker 600 MHz).Chemical shifts were expressed in δ (ppm) (Appendices 1 and 2).

Microorganisms
The Gram positive (Staphylococcus aureus KZN) and Gram negative (Salmonella spp.KZN, Escherichia coli ATCC 8739, Pseudomonas aeruginosa ATCC 19582, Proteus mirabilis KZN) bacteria were obtained from the Department of Microbiology, University of Zululand.Antibiotic resistant strains of clinical isolates were obtained from the Lancet Pathology Laboratory (Durban, South Africa).Mueller-Hinton agar (Merck Catalogue No. 1.05435.0500)was used to maintain stock cultures and these were kept at 4°C.

Disk diffusion method
Antimicrobial activity of the triterpenes was investigated by the agar disc diffusion method (Vlietinck et al., 1995).Overnight bacterial cultures were diluted to a final cell density (1.0 × 10 8 CFU/ml) equivalent to 0.5 McFarland standard.Sterile paper discs (6 mm diameter), impregnated with the triterpene (20 mg/ml 10% DMSO) were placed on nutrient agar which was inoculated with bacterial suspension (1.0 × 10 8 CFU/ml).Dimethyl sulphoxide (DMSO) (10%) was used as negative control while ampicillin and neomycin were used as positive controls.The inoculated plates were incubated at 37°C for 24 h.The antimicrobial activity was evaluated by measuring the zone of inhibition (mm) against the test organism.

Minimum inhibitory concentration (MIC) and Minimum bacterial concentration (MBC)
The method previously described by Eloff (1998) was adopted to determine the MIC of the triterpenes.Overnight bacterial cultures in Muller-Hinton broth were standardized to 0.5 McFarland standard and 96-well plates were used to quantitatively determine the MIC and MBC of the compounds.The tests were replicated thrice and the mean values reported.Nutrients broth (50 μl) was added to all wells of the 96-well plate and 50 μl of the triterpene (20 mg/ml, in 10% DMSO) was introduced to the wells in the first row (A) and mixed well.Sample mixture (50 μl) was removed from all the wells in the row A to perform a 2-fold serial dilution down the rows.The last 50 μl was discarded.Respective bacterial cultures (50 μl) were introduced into the corresponding wells.DMSO (10%) was used as negative control while ampicillin and neomycin were used as positive controls.The plates were covered and then incubated at 37°C for 24 h.Iodonitrotetrazolium chloride (INT) (20 μl, 0.2 mg/ml) was added to all of the wells and the plates were then further incubated at 37°C for 30 min.The MIC was recorded as the lowest concentration of the tested compounds at which no visible microbial growth was observed.The MBC of the triterpenes was determined by removing 10 μl of each culture medium from the wells that no bacterial growth was observed onto sterile nutrient agar plates.The plates were then incubated at 37°C for 24 h, after which they were observed for presence or absence of growth.The MBC was recorded as the minimum concentration at which no bacterial growth recurred.Ampicillin and neomycin were used as positive controls.

Lactate dehydrogenase (LDH) release assay
The effect of the triterpene on the bacterial membrane integrity was evaluated by using the cytosolic LDH release assay (Tadić et al., 2012).Fresh cultures of the most susceptible bacteria were treated and incubated (at 37°C for 24 h) with the MBC of the compound.The bacterial suspension was centrifuged at 5000 × g for 5 min.Supernatant (100 μl) was collected and mixed with 100 μl of reaction mixture (54 mM lactic acid, 0.28 mM of phenazinemethosulfate, 0.66 mM INT, 1.3 mM NAD + ).The mixture was incubated at 37°C for 10 min.DMSO (10%) was used as negative control and 3% triton X-100 was used as positive control.The pyruvate-mediated reduction of INT into highly-coloured formazan (red) was measured at 490 nm using BioTek plate reader (ELx 808 UI, Biotek Instrument Supplies).The experiment was replicated three times and the mean values reported.The amount of LDH (%) released upon cellular loss of membrane integrity was calculated using the formula: Where AE-absorbance of test compound-treated cell culture, ACabsorbance of control (cell medium alone) and AT-absorbance of Triton X-100 lysed cells [representing maximal (100%) LDH released].

Bacterial DNA damage
The method described by Liu et al. (2011) was adapted to determine the effect of the triterpenes on bacterial DNA.This was determined on the bacteria that were most susceptible to the compounds.Fresh bacterial cultures were treated with the MBC of the compound.Both treated and untreated bacteria were incubated at 37°C for 24 h.Bacterial DNA was extracted and purified using ZR Fungal/bacterial DNA MiniPrep™ kit (Zymo Research, USA, Catalogue No. D6005).Briefly, bacterial samples were added directly to ZR lysis tubes and centrifuged at 10 000 × g for 10 min.
The pellet was collected and resuspended in the lysis solution for 5 min.Fungal/bacterial DNA binding buffer was then added to the suspension.This was centrifuged at 10,000 × g for 10 min and supernatant was collected.The bacterial DNA was precipitated following a series of centrifugation and washing in buffers.Pure DNA was then eluted with the elution buffer.Agarose gel electrophoresis (150 v for 30 min) was used to evaluate damage of both untreated and triterpene-treated DNA.This was run along with DNA maker.The DNA was visualized using a vilberlourmate Gel documentation system.

Structural elucidation and characterization
The structures of the isolated compounds (Figure 1) were established and confirmed through 1 H and 13 C NMR.The physical and spectral data of 3β-hydroxylanosta-9,24dien-21-oic acid (1) have been previously described (Mosa et al., 2011).The IR spectrum showed absorption bands for hydroxyl (3360, 2581 cm -1 ) and carbonyl (1702 The results are expressed as mean ± SD.Compd-Compound, Clox-Cloxacillin, Cipro-Ciprofloxacin, Levo-Levofloxacin, Meth-Methicillin, Oxa-Oxacillin. cm -1 ) functional groups which further confirmed the structure.The 1 H-NMR of the compound (2) followed the same triterpenoid pattern with a large clusters of signals of CH 3 , CH 2 and CH between δ H 2.5 and 0.8 observed in 3β-hydroxylanosta-9,24-dien-21-oic acid (Mosa et al., 2011).The 13 C-NMR of this compound was also similar to that of 3β-hydroxylanosta-9,24-dien-21-oic acid, with the presence of four olefinic carbon atoms between 145 to 118 ppm, and five quaternary carbon atoms confirming the lanosteryl skeletal structure.The presence of an ester carbon atom at δ C 177.3 instead of a carboxylic carbon at δ C 181.5 suggested that this compound is the methyl ester of 3β-hydroxylanosta-9,24-dien-21-oic acid.Table 2 presents a detailed assignment of the 13 C-NMR and significant 1 H-NMR of the triterpene.The absorption bands for hydroxyl (3469 cm -1 ) and carbonyl (1683 cm -1 ) functional groups observed on IR spectrum also further assisted in confirming the NMR structure.

Biological activity
Plants have always been an untapped source to provide structurally diverse bioactive compounds as potential therapeutic agents, including antimicrobials.The antibacterial activity of the triterpenes from P. longifolia is shown in Tables 2 to 4. The two triterpenes exhibited antibacterial activity against both Gram negative and Gram positive bacteria with the MIC and MBC values ranging from 0.16 to 5.00 mg/ml and 0.63 to 5.00 mg/ml, respectively.It is noteworthy that the triterpenes were active against the antibiotic resistant clinical isolates; the P. aeruginosa and Staphylococcus spp.being the most susceptible organisms.Triterpenoids from Momordica balsamina (Ramalhete et al., 2011), Alisma orientale (Jin et al., 2012) and Carpobrotus edulis (Martins et al., 2011) also exhibited antibacterial activity on various resistant bacterial strains including the Staphylococcus spp.Popova et al. (2009) also reported the antibacterial activity of lanostane triterpenes from wood-decay fungus Fomitopsis rosea against S. aureus.The ability of both compounds to exhibit strong activity even on the resistant bacterial strains indicates their potential to be developed into effective antimicrobial agents.
Antimicrobial drugs exert their therapeutic activity through various mechanisms which include inhibition of proteins, cell wall, cell membrane and nucleic acid synthesis (Riaz et al., 2011).The antimicrobial activity of terpenoids has been associated with among others, bacterial cell membrane disruption by the lipophilic compounds (Saleem et al., 2010).The evaluation of the bacterial DNA damaging effect of the triterpenes indicated that the investigated triterpenes did not exhibit any DNA damaging effect.DNA protective effect of some triterpenes has previously been reported (Ramos et al., 2010;Smina et al., 2011).The cytosolic lactate dehydrogenase (LDH) release assay was used to evaluate the effect of the compound 2 (compound 1 was not tested due sample limitation) on the bacterial cell membrane integrity.The release of the cytosolic LDH into the extracellular medium is indicative of the cell membrane damaging effect of the triterpene.It is noteworthy that despite the complex Gram negative bacteria cell wall, the highest percentage LDH release was observed in case of P. mirabilis (89.6%) and E. coli (76.3%) (Table 5).While some compounds act by damaging both membrane and DNA (Liu et al., 2011), it is apparent that the triterpenes exert their antibacterial activity by affecting the microbial cell membrane integrity rather than damaging DNA.

Table 1 .
13C-NMR data and significant 1 H-NMR data of compound 2. Chemical shifts are expressed in δ (ppm).

Table 2 .
Zones of inhibition (mm) of the triterpenes on some sensitive and antibiotic resistant bacteria.

Table 3 .
MIC and MBC in mg/ml of the triterpenes on the general bacteria.