Antibacterial Activity of Clinacanthus nutans Polar and Non-Polar Leaves and Stem Extracts

1Health Sciences Division, Abu Dhabi Women’s College, Higher Colleges of Technology, Abu Dhabi, United Arab Emirates, 41012. 2Department of Pharmaceutical Chemistry, School of Pharmacy, International Medical University, Kuala Lumpur, Malaysia, 57000. 3Centre for Cancer and Stem Cells Research, Institute for Research, Development and Innovation, International Medical University, Kuala Lumpur, Malaysia, 57000. *Corresponding Author E-mail: lerin@hct.ac.ae

Despite more aggressive antimicrobial stewardship being promoted, the evolution of bacterial infections fueled by excessive antibiotic pressure is still a pressing problem in the clinical setting 1 . Due to limitations in the development of new antibiotics, researchers are turning to natural products to aid in drug discovery [2][3] .
Clinacanthus nutans in the Acanthaceae family, also known as 'belalai gajah' is grown in tropical weathered countries in Asia such as in Malaysia, Thailand and China 4 . C. nutans was used traditionally by Asians as an antifungal 5 , cytotoxic 6 , anti-inflammatory 7 , antioxidant 8 , and anti-pyretic 9 agent. In addition, C. nutans had been used to treat certain diseases like skin rash, diabetes and insect bites 10 . Traditionally, the dried leaves of C. nutans had been mixed with fresh juices or green tea, or the leaves are put directly on the infected skin with nettle rashes. Fazil et al. (2016) 9 and Mai et al. (2009) 11 described the antiproliferative effects of flavonoids from C. nutans fresh and dried leaves against lung cancer cells, Both, the fresh and the dried leaves possessed similar effect 9,11 . In addition, Alam et al. (2016) 12 proved that the stem of C. nutans showed a wide range of properties such as anti-viral and anti-inflammatory activities extracted from ethanol 80%, while the leaves show a broad cytological activity,cholinergic modulation and anti-papillomavirus infectivity from petroleum ether extract 12 . C. nutans leaves showed an inhibition effect on fungi like P. oryzae 5 , and antibacterial activity on the gram-positivebacteria was greater than the gram-negative bacteria; this study was done using stem and leaves (crude extracts) on three bacteria, that are Bacillus cereus, Escherichia coli and Salmonella enterica 6 .
Our previous study identified that the polar leaves C. nutans extracts exhibited anti-inflammatory effects by inhibiting the lipopolysaccharides(LPS) induced Toll-like receptor-4 (TLR-4) activation, nitric oxide accumulation, inflammatory cytokines reduction and TLR-4 related inflammatory proteins expressions 13 . Therefore, in our study, we will investigate the antibacterial effect in gram-positive and gram-negative bacteria using the polar and non-polar stem and leaves extracts.

Bacteria
The

Preparation of the extracts
The plant was collected from previous study and extracted using the same procedure 13 . Briefly, the plant was dried, powdered and separated into leaves and stem bark. The powdered stem and leaves were extracted with non-polar solvents (diethyl ether and hexane) or polar solvents (dichloromethane and methanol) by immersion itin the solvent at room temperature for three days. Four extracts were prepared, namely non-polar leave extracts (LN), polar leave extracts (LP), non-polar stem extracts (SN) and polar stem extracts (SP). The extracts were filtered and then we removed the solvents by rotary evaporator at 60°C. The concentrations of each extract used for testing were 0.25, 0.5, 1, 2, 4, 8, 16, 32 mg/mL.

Broth Microdilution test
The broth microdilution method was used to help to determine the minimum inhibitory concentration (MIC). Testing was carried out based on the CLSI M07-A8 guidelines 14 , but with some changes to aid visualization: resazurin (7-hydroxy-3H-phenoxazin-3-one 10-oxide) (Sigma Aldrich, USA) was added to a final concentration of 0.002%. In summary, dilutions were done in triplicate, each well containing 40µL of bacterial suspension (final concentration of 2-8 x 10 5 cfu/mL) with 50µL of test compound that inoculated with 10µL of resazurin. Gentamicin (Bioworld, USA), at 32µg/ mL for the highest concentration (Oxoid, UK) and DMSO at 5% at highest concentration were used as negative and positive controls respectively. Disks were obtained from Oxoid, UK. The plates were incubated for 18h at 37°C. The MIC value was taken at the lowest concentration at the resazurin color change from purple to pink or colorless indicating the reduction in the growth of the bacteria, resazurin (7-Hydroxy-3H-phenoxazin-3-one 10-oxide). Pink demonstrates bacteria that are alive, while the blue demonstrates the death of bacteria.

statistical analysis
All data were presented as mean ± standard deviation (S.D.) from at least three independent experiments. SPSS (version 18.0) for Windows was used to conduct the one way Analysis of Variance (ANOVA) post hoc Dunnett' t-test, in which statistically significant was defined as p-value was not more than 0.05.

results and discussion
All four extracts from C. nutans showed growth inhibition in all twelve bacteria species as extract concentration increased ( Figure 1). LN had the strongest inhibitory effect in all bacteria compared to LP and SP, as well as SN. Figure  Fig. 1. The viability percentage of twelve bacteria species under different concentrations of LN, LP, SN and SP extracts of C. nutans. Statistical significance difference compared to vehicle control is indicated as * p < 0.05 1 shows the effects of different antibiotic's concentrations extracted from the C. nutans LN on twelve species of bacteria. The effective concentration ranged between 0.25 mg/mL which was the lowest concentration and 1 mg/mL.For example, P. mirabilis viability was dropped to 41% while P. mirabilis and K. pneumoniae dropped to slightly above 30%. As for Enterococcus faecalis, it dropped to only 20% viability. E. coli being the most effected showed viability at percentage of 5% at 16mg/mL, followed by P. vulgaris with 7.18% and B. subtilis with 8.16%. Lastly, the viability percentage of all the bacteria species increased slightly when the concentration of the antimicrobial doubled to 32 mg/mL. Table 1 represents the MIC 50 and MIC 90 values in mg/mL of LN extracted from C.nutans.
For the LP extracts, a dose-response viability rate was observed in twelve species of bacteria. Figure 1 compares the viability percentage changes after treating the bacteria with different concentrations of LP extracts. Concentration levels of 0.25 to 4 mg/mL show an almost constant viability percentage of 81% for S. aureus. However, the percentage fell noticeably from 81% to 20.26% at 16 mg/mL.Bacterial species like S. epidermidis, B. subtilis, E. coli, P. aeruginosa showed a drop in viability percentage when antimicrobial concentration increased from 4 to 6 mg/mL. However, for the bacteria species like E. faecalis, P. mirabilis, K. pneumoniae and P. vulgaris their viability percentage decrease when the concentration of the antimicrobial increased from 8 to 16 mg/mL.B. subtilis shows to be the most effected by the treatment compared to the others, the viability showed an early drop in percentage, it was 141% at 2mg/mL and reached a value of 26% at 16 mg/mL. Table 1 shows the MIC 50 and MIC 90 values in mg/mL of LP extracted from C.nutans.
For the activity of SN extract, the reduction of growth was not significant for all bacteria tested compared to other extracts ( Figure  1). Table 2 shows the MIC 50 and MIC 90 values in mg/mL of SN extracted from C.nutans. The highest reduction of viability was to 62.73% at 32mg/ mL for P. vulgaris which is higher than the other extracts. The polar stem was more effective than non-polar, because the highest reduction in growth compared to non-polar stem was to 12% at 32mg/ mL for E. coli.The growth of B. cereus was low in 0.25 to1 mg/mL but then it peaks at 2 mg/mL to 209% and then sharply went down at 4 mg/mL to 83%. Table 2 represents the MIC 50 and MIC 90 values in mg/mL of SP extracted from C.nutans.
In the present study, leaves and stems extracts obtained from C. nutans were evaluated as an antimicrobial agent against twelve bacteria. The C. nutans is a herb known for its medical properties. This plant grows in a tropical climate, in countries like Malaysia and Thailand. The C. nutans therapeutic characteristics has spurred the interest and attention of many researchers. This medical herb has numerous chemical compounds that possess antibacterial activities such as, â-sitosterol, stigmasterol and flavonoids 15 . The population of South East Asia traditionally used this plant to treat various medical conditions such asinsect bites, skin rashes, herpes infection, inflammation, and cancer. Moreover, the C. nutans plant has been shown to possess antimicrobial activity. Thus, it is used to treat various skin conditions that are caused by bacterial colonization in Thai traditional medicine 16 .
The results of our experiment showed that leaves extract was more potent antimicrobial compared to stems at the validity of 5% in concentration of 16 mg/mL. In a past study, Justiciaadhatoda from the Acanthaceae family species was tested for the antimicrobial effects of the methanolic and leaf extract of the plant. The results indicated that the antimicrobial effects of the leaf extract surpassed the methanol extract 17 . Moreover, the same study was conducted on Crossandra infundibuliformis (Acanthaceae family), and the results illustrated that the ethanol extracted from the leaf of the plant has better antibacterial activity compared to others 18 . Hence, this confirmed that the leaf extracted from C. nutans is stronger than the stems since all plants are from the same family.
The non-polar extracts overall indicated a more effective antimicrobial effect compared to polar extract at validity of 5% in concentration of 16 mg/mL.In a past study, the non-polar extracts showed more effective antimicrobial activity against gram-positive bacteria 19 . In addition, another study showed that chloroform is the best solvent that has been extracted from non-polar solvents from Angelica archanglica for biological activity 20 .
In our study, the antimicrobial activity of C. nutans extracts was more effective against the gram-negative bacteria than the gram-positive. In fact, the inhibitory results found against gramnegative bacteria were unexpected, due to the fact that usually they are more resistant compared to gram-positive bacteria. In general, the cell wall of the gram-positive bacteria mainly consists of peptidoglycan, and it represents 90% of the cell wall. On the other hand, the gram-negative bacteria have a more complex cell wall. Similarly, to the gram-positive bacteria, the gram-negative bacteria cell wall is composed of peptidoglycan. However, peptidoglycan represents 5-10% of the total cell wall. In addition, the gram-negative bacteria have an outer layer that is known as (LPS) 21 . C. nutans extracts may possibly have more effect to LPS than the peptidoglycan. However, Andrographis paniculata (Acanthaceae family), a plant from the same family as C.nutans showed antibacterial effects towards gram-positive bacteria. This may be because the mesh-like peptidoglycan layer is more accessible to the extracts which permeates the cell wall. The outer membrane of the gramnegative cell wall may contribute to their resistance against C. nutans as the membrane is composed of lipopolysaccharide which restricts entry of the plant extract.

conclusion
In this study, the results revealed that LN of C. nutans have a stronger antibacterial activity than LP, NS and NP. The gram-negative bacteria were more sensitive to the extracts compared to gram-positive bacteria. Due to limited studies on C. nutans extracts, further studies must be conducted to elucidate the pharmacologically active compounds from this plant. This may lead to the development of a new generation of antibiotics.