Synthesis, Characterization and In vitro Antibacterial Activity of A Novel Strigolactones Analogues TIT3

Traditional antibiotic abuse caused increased the incidence of bacteria resistance to treatment. Strigolactones (SLs) are phytohormones that play a vital role in the plant growing root, shoot and flowering. We previously reported that, SLs analogues exert pro-apoptotic effects on HepG2 cell lines. The current study investigated the antimicrobial activity of selected SLs analogue TIT3 against different strains of bacteria including gram positive and gram negative bacteria: Staphylococcus aureus, Salmonella typhimuriu, Escherichia coli, Klebsiella pneumoni and B. subtilis. The results obtained were compared with 100 μg amoxicillin. Results obtained showed that, TIT3 inhibited the growth of S. cereus (50 μg), Salmonella typhimuriu (20 μg), Escherichia coli (30μg), Klebsiella pneumoni (50 μg) and B. subtilis (50 μg) with mean inhibition zones diameter being 12.1 mm , 13.2 mm, 12.5, 8.9 and 12.9 mm respectively. It was concluded that, TIT3 is promising effective antibacterial agents compared with amoxicillin for different strains of bacteria that resistant to different antibiotics.


iNtRODUCtiON
Traditional antibiotic abuse caused increased the incidence of bacteria resistance to treatment .Bacteria that resist to major antibiotics have ability to grow and transform to dangerous species. Control of infections induced by multidrug-resistant species is a challenge obstacle for hospitals 1 . In Clinics, patients suffered from these bacteria need more time and much cost for relief 2 . Development of a novel antibacterial compounds with high efficacy from medicinal plants are suitable alternative sources. Microbial resistance to chemotherapeutic by antibiotics is considered as one of the most problems in medicine 3 . This resistance is an event of the adaption of pathogenic bacteria to antibiotics used in hospital 4,5 . Some microbes have developed resistance to all types of antibiotics and these multidrug-resistant microbes are dangerous to invade different tissues 6 . In order to design a novel antibiotic with different mechanism of action, plants are considered as a main sources for new bioactive compounds with high efficacy. Phytochemical are vital sources of compounds against diverse organisms including fungi, yeasts and bacteria 7 . It exerts its activity by inhibiting cell membrane synthesis, modify bacterial permeability and sensitivity.
However, only 35 percent of the microbial infection have been treated using synthetic or derivatives products. This is due to resistant of pathogens for prolong use of antibiotics [8]. The resistance to antibiotics increased dramatically due to mutation of microbial genome. To decrease the microbial resistance to antibiotics is by explore a novel antibiotic resistance inhibitors produced from plant origin 9 . It was found that, plants possess ability to protect themselves against different pathogens by producing a variety of phytohormones. Medicinal plants are widely used as complementary medicine. Due to their safety to human phytomedicines are used as therapeutic index for the exploring novel drugs. It can be used as anticancer, antioxidants and antiinflammatory 10 .
Strigolactones (SLs) are a novel class of phytohormones synthesized by different plant species 10 . The SLs structure consists fused-ring system connected via an enol ether bridge 11 . Previous study reported that, SLs exert its effect by regulating different parts in plant development including shoot and roots 12 . It has been reported that, SLs analogs stimulate cell cycle phases arrest and programme cell death in different types of human cancer 13 . It was found that, growth inhibition of normal cells treated with SLs analogs. In other study, inhibition was observed in some cancer cells cell lines 14 .
Moreover, SLs showed as new anticancer agent in breast cancer 15 . SLs analogs showed down-regulation of proteins involved in cell cycle (cyclin) 16 . These facts proposed that SLs are potent and promising anti-cancer activity. SLs enhance induction of G2 arrest, stimulate apoptosis and suppress viability 17 . The bacteria is protected by thick cell wall that composed of layers of peptidoglycan, cross-linked covalently to form polymer of peptide-linked β-(1-4)-N-acetyl glucosamines. The mechanical strength of the cell wall is important for bacterium's ability to survive under vigorous environmental conditions as osmotic pressures. The degree of cross-linking can be correlated with the structural integrity of the cell. It was reported that, transglycosylase and transpeptidase enzymes, which add disaccharide pentapeptides contribute to peptidoglycan formation and cell wall integrity 18 . In order to contribute to explore a novel SLs analogues (TIT3) as antimicrobial activity against different pathogenic bacteria. This study investigated the antimicrobial activity of TIT3 against different specis of bacteria as Staphylococcus aureus, Salmonella typhimuriu, Escherichia coli, Klebsiella pneumoni and B. subtilis.

Synthesis of Strigolactons
The SLs (TIT3) was synthesized and identified by Prof. Tadao Asami (University of Tokyo, Japan). We previously mentioned the details of synthesis and published recently 19 . Working standard dilutions to 10 µM was done using DMSO, the concentration of DMSO was maintained at 0.1-0.5 %. The structure of tested SLs, as TIT3 with their molecular weights was shown in (Fig. 1) compared with amoxicillin as positive control (Fig. 1b).

determination of MIC
Antimicrobial activity of the TIT3 at different concentrations were tested against different bacterial strains including Staphylococcus aureus, Salmonella typhimuriu, Escherichia coli, Klebsiella pneumoni and B. subtilis. The bacteria samples were obtained from Microbiology Department, Faculty of Science, KAU . Growth standard media was used (Per one liter media,1 g glucose, 15 g peptone, 6 g Sodium chloride) at pH 7.0. The antimicrobial activity of TIT3 were tested by using 10 μl of each type was added to sterile test tube contain 10 ml nutrient broth. Blank and negative control samples were done without microbe. After 24 h, growth was visually assessed by changes in turbidity and quantified by measuring O.D at 600 nm (Shimadzu spectrophotometer). Amoxiciilin (100 µg/ml) was used as a positive control. The diameters of growth inhibition zones was measured in mm. The initial screen to determine MIC of TIT3 by adding different concentration (0 -100 μg). Each concentration was incubated an overnight with media. After 24 h, growth was visually assessed as mentioned above.

Bioassay the cytotoxicity of TIT3by using brine shrimp lethality test
The toxicity of TIT3 was determined according to Michael et al. 17 at different concentrations from (10-1000 μg/mL in seawater containing 2% DMSO (v/v). Ten shrimps larvae was used in each test, three replica each test. A positive control from potassium dichromate solution (LC 50 = 20-50 μg) was tested compared to blank test. The survivor count was recorded after 24 hours by using microscope and the mortality percent was calculated. Cytotoxicity was significant if the LC 50 < 200 μg/ml.

ResUlts
Bioassy lethality activity of the TIT3 was shown in Table 1. It was found that 30% mortality at 500 μg/ml concentration and its LC 50 was 400 μg/ml which was considered moderately toxic. The Standard potassium dichromate revealed LC 50 was 200 μg/ml. However DMSO showed no mortility.
Data obtained (Table 2) indicated that TIT3 had variable activity against different species. It was found that, the maximum activity against S. aureus at 50 µg/ml, Salmonella typhimurium at 20 µg /ml, Escherichia coli at 30 µg /ml and Bacillus cereusat 50 µg /ml.
We screened different concentrations of three SLs analogues TIT3, (0-100 µg). Results obtained in Fig.s (2-6) showed that, TIT3 at concentrations 50 and 20 µg showed the most effective inhibitory effect on s.auresu and B. subtilis respectively with inhibitory effect against E. coli >80% and 77% inhibition for B. subtilis respectively. It is clear that, above these concentrations showed decreased activity.

DisCUssiON
The cytotoxicity of TIT3 was investigated on shrimp larvae. The data obtained in (Table 1) showed that by at very high concentration and exposure the toxicity increased. Recent published work by our research team Hasan et al. 19 indicated that, TIT3 was effective on HepG2 cell line but more save of normal cell caused early apoptotic cell. For that, TIT3 is more save for eukaryotic cells. The MIC was similar to ampicilline for both Grampositive and Gram-negative bacteria.
To examine the antimicrobial potential of the different concentrations of TIT3 . Overall, the results obtained revealed that TIT3 induced inhibitory effects against different species of bacteria with different efficacy. We found that by increasing the concentrations of TIT3, in making it more efficient in inhibiting efficacy. This effect is most pronounced in the case of S. aureus at, where the higher concentration is clearly associated with higher inhibition. The antibacterial properties of the TIT3 may be due to interference with cell wall synthesis of bacteria, so inhibiting its growth. TIT3 showed a better antibacterial activity through inhibition of bacterial several enzymes, microbial adhesion, and cell envelope transport proteins 20 . The antibacterial potential of TIT3 was found to be maximum against Salmonella typhimurium at low concentration. This antimicrobial susceptibility reported promising new vision for development and production of a novel agents against different pathogens. Thus MIC determined reflecting that TIT3 has antibacterial potential and that it gives real concentration required to inhibit bacterial growth. In conclusion, the TIT3 was found to be more effective against B.subtilis and S. cerevisiae. The potent activity may be due to the penetrating power of TIT3 to bacterial cell.