Brucellosis, a bacterial zoonosis and major public health concern due to its high morbidity rate. The prevalence of infection in humans is directly associated with occurrence in animals, particularly in domestic ruminants.1 Among Brucella species B. melitensis, B. abortus and B. suis are pathogenic for humans. While brucellosis occurs worldwide, it is endemic in the Mediterranean basin, the Middle East, Western Asia, Africa and Latin America.2
Infection of brucellosis causes significant economic losses by comparatively low milk production in livestock, abortion, weak off-springs, public health and international trade implications.3 The real rate is estimated to be 10 to 25 times more than annual reports.4Brucella is non-motile, small, gram negative, non-spore forming, and strictly aerobic coccobacilli. It is mostly positive for catalase and oxidase tests and shows various results in urease tests.5 Brucellae genus shows little variation genetically, presently eleven Brucella species have been recognized, they are genetically very similar although each have different host preferences.6 Brucellae are highly potent pathogen in animals, humans and also effective biological agents for use in biological weapons even at very low concentration of 10 bacteria. Brucellae are easily transmitted to humans via aerosols and these make bacteria most attractive for defence researchers.7
Infectious diseases pose a severe health concern worldwide. The development of drug resistant pathogens due to haphazard use of antibiotics has increased the need for new source of antimicrobial agents. This has encouraged screening of new plant species for potential medicinal and antioxidant properties.8,9 In general, the Gram-negative bacteria show less sensitivity to plant extracts possibly as a result of their extra lipopolysaccharide and protein cell wall that provides a permeability barrier to the antibacterial agent.10 Furthermore, the Gram-positive bacteria are more sensitive to the plant extracts because of the single layer of their cell wall, while the double membrane of Gram- negative bacteria should make them less sensitive.11
Medicinal plants have been recognized as a part of the evolution of human healthcare for thousands of years. Medicinal components from plants play an important role in traditional as well as in modern medicine. Antimicrobial resistance is progressively becoming a serious threat to global public health. According to World Health Organization (WHO) report on antimicrobial resistance in 2014, overcoming the antibiotic resistance is the major challenge for the next millennium.12 Screening of plants for antimicrobial agents has gained importance, because WHO is encouraging and promoting the development and utilization of medicinal plant resources in the traditional system of medicine.
The usage of herbal plants as traditional health remedies is the most preferable by 80% of the world population in Asia, Latin America and Africa and has been reported to have minimal side effects.13 For treatment of brucellosis, a combination of antibiotics that penetrates the macrophage should be used. The choice treatment for human brucellosis caused by B. melitensis field strains is a combination of long-acting tetracyclines and streptomycin. Additionally, studies have shown that for treatment of patients with B. melitensis vaccine strains Rev1, a gentamicin/doxycycline combination may be the first choice.1 In general, tetracycline/aminoglycoside combinations are the most common antibiotics used for brucellosis treatment. However, because of high rates of treatment failure or relapses due to emerging resistance, the treatment of brucellosis is still problematic. Thus, new antibacterial compounds are becoming necessary for brucellosis treatment. Medicinal plants have always been sources for new drug discovery. Plants readily synthesize substances for their defence against insects, herbivores, and micro organisms.14 Moreover, they might produce secondary antimicrobial metabolites as a part of their normal growth and development or in response to stress.15
Hence the objective of the study is to screen for antibrucellosis activity of ethonolic extracts of Acacia nilotica, Withania somnifera, Eugenia jambolana, Callistemon citrinus, Clerodendrum inerme, Terminalia arjuna, Thevetia peruviana, Leucas aspera, Hemidesmus indicus, Gloriosa superba, Cymbopogon citrates, Acorus calamus, Cinnamon, Thuja occidentalis and Santhalum album against antibrucellosis activity in vitro. Purification of crude ethanolic plant extract by TLC profiling and identification of the bioactive metabolites by GC-MS.
MATERIALS AND METHODS
Bacterial reference strains
Brucella strains -Brucella abortus, Brucella melitensis, Brucella suis were procured from Indian Veterinary Research Institute (IVRI), Izatnagar, Bareilly, Uttar Pradesh, India. They were tested for the purity, biochemical and molecular characteristics before use. Type III Biosafety containment was used to culture the bacteria. Escherichia coli (1610), Salmonella typhimurium (98) Bacillus cereus (430), Shigella flexneri (1457), Vibrio cholera (3904), Pseudomonas aeruginosa (1688), Vibrio paraheamolyticus (451), Bacillus subtilis (6939) and Enterobacter aerogenes (13048) bacterial strains were obtained from Microbial Typing Culture Collection (MTCC), Chandigarh, India and American Tissue Culture Collection (ATCC) and were cultured as per the protocol prescribed by MTCC and ATCC respectively.
Plant collection and identification
Different medicinal plants were collected from Western Ghats of Karnataka, India. The taxonomic identification of these plants was done by Prof. G. R. Shivamurthy, former professor, Department of Botany, University of Mysore, Karnataka, India.
Plant materials collection and processing
The plant leaves were thoroughly washed with tap water to remove dusts and other unwanted materials accumulated on the leaves from their natural environment. The dust free leaves were allowed to dry under shade in the laboratory for 20 days. The dried leaves were powdered by using electric blender. Finally, fine powder was collected from the powdered leaves by sieving through the muslin cloth and used for extraction.
Twenty gram of powdered plant material was put in a 200 mL conical flask and 100 mL of ethanol solvent was added. Conical flask was covered with aluminium foil and kept in a reciprocating shaker for 24 h for continuous agitation at 130 rev/min for thorough mixing and also complete extraction of active materials to dissolve in the solvent. Then, extract was filtered by using muslin cloth followed by Whatman No 1 filter paper and finally the solvent from the extract was removed by using rotary vacuum evaporator at water bath temperature of 50°C. Finally, the residues were collected and used for the experiment.
Antibacterial susceptibility assay
The test isolate was grown in Muller-Hinton Broth (Merck, USA) medium at 37 °C for 22 h. Final inoculum bacterial numbers were adjusted to 108 CFU/ml. A total of 0.1 ml of bacterial suspension was poured on each plate containing Muller-Hinton Agar (MHA). The lawn culture was prepared by sterile cotton swab and allowed to remain in contact for 1 min. Different concentrations of ethanolic extracts (1, 5, 10, 25, 50, 100, 200, and 300 mg/ml) from each plant were prepared. The sterile filter paper discs (6-mm diameter) were saturated by 50 μl of different concentrations of each extract and then were placed on lawn cultures.16,17 The Petri dishes were subsequently incubated at 37°C for 24 h and the inhibition zone around each disc was measured in mm. As positive controls, discs (Difco, USA) containing streptomycin 10 μg, gentamicin 10 μg and Ciprafloxin 10 μg were used. Further the TLC profiling was carried out for the extract with strongest antibrucellosis activity.
Thin Layer Chromatography profiling (TLC)
TLC system equipped with a sample applicator was used for application of samples. Five ul of leaf ethanol extracts was separately applied on 5 × 10 cm chromatographic pre-coated silica gel plates (TLC grade, Merck, USA) as the stationary phase. The TLC plates were developed in a twin trough glass chamber containing mixture of chloroform and methanol (99: 1 v/v) as the mobile phase. The plates were removed when the solvent front has moved to the defined level, subsequently allowed to dry. After drying, the spots on the developed plates were visualized under visible (white), short UV (254 nm), and long UV (366 nm) light. Extract was expressed by its retention factor (Rf). Values were calculated for each spot using the following formula:
Preparative TLC was carried out to isolate the separated compounds based on Rf values was done to obtain substantial quantities for antimicrobial test.
Minimum inhibitory concentration MIC
The test isolate was grown in Muller-Hinton Broth (Merck, USA) medium at 37°C for 22 h. Final inoculum bacterial numbers were adjusted to 108 CFU/ml. A total of 0.1 ml of bacterial suspension was poured on each plate containing Muller-Hinton Agar. The lawn culture was prepared by sterile cotton swab and allowed to remain in contact for 1 min. Different concentrations of TLC purified ethanolic extracts (25, 50, 100, 200, 400, 800 and 1200 μg/ml) from each plant were prepared. The sterile filter paper discs (6-mm diameter) were saturated by 50 μl of different concentrations of each extract and then were placed on lawn cultures.16,17 The Petri dishes were subsequently incubated at 37°C for 48 to 72 h anaerobically and the inhibition zone around each disc was measured in mm. As positive controls, discs (Difco, USA) containing gentamicin 10 μg.
Gas chromatography-mass spectrometry
A Hewlett-Packard 5890 Series II Chromatograph equipped with a FID detector and HP-2 fused silica columns (25 m × 0.32 mm, 0.25 μm film thicknesses) was used. The samples, dissolved in hexane, were injected in the split less mode into helium carrier gas. Injector and detector temperatures were maintained at 250°C. The column temperature was programmed from 60°C (after 2 min) to 220°C at 4°C/min, and the final temperature was held for 20 min. Peak areas and retention times were measured by electronic integration of by computer. The relative amounts of individual components are based on the peak areas obtained, without FID response factor correction. GC-MS analyses were carried out on a Hewlett-Packard 5970A mass selective detector (MSD), directly coupled to HP 5790A gas chromatograph. A 26 m × 0.22 mm column, coated with 0.13 μm of CP-Sil 5CB was employed, using helium carrier gas. The oven temperature program was 60°C (3 min), then 5°C/min to 250°C (30 min). Other conditions were the same as described under GC. Electron ionization (EI) mass spectra were acquired over a mass range of 10-400 Da at a rate of 2/s.
Identification of the compounds
The identification of the compounds present in the TLC purified ethanolic extracts were based on direct comparison of the retention times and mass spectral data with those for standard compounds, and by computer matching with the Wiley 229, Nist MS Library.
The antibrucellosis activity was evaluated for different ethnomedicinal plants using disc diffusion method and represented in table (Table 1). C. citrinus showed excellent biocidal activity against B. abortus B. melitensis and B. suis, moderate activity was displayed by A. nelotica against B. abortus, while T. arjuna exhibited negligible activity against B. abortus and B. suis. Among the plant sources crude ethanolic C. citrinus (Figure 1) showed dose dependent inhibition against Brucella spp. such as B. abortus, B. melitensis and B. suis. The MIC concentration was observed according to Clinical and Laboratory Standards Institute (CLSI) for B. abortus (1.5 mm) at 300 mg/ml concentration, B. melitensis (1.7 mm) at 300 mg/ml and B. suis (1.7 mm) at 100 mg/ml concentration. This results were compared to the standard SM, GN, CIP –1.4 1.6, 1.3 respectively in the present study. The statistical significant was observed for brucella spp. P≤ 0.05 for the B. Suis, P≤ 0.05 for B. melitensis and P≤ 0.01 for B. abortus
Thin layer chromatography
The TLC plate was developed in respective mobile phase chloroformmethanol (99:1, v/v) for separations of C. citrinus ethanolic extract bioactive compound (Figure 3), about over 8.5 cm, resulted in four bands, with four spots, spot A Rf value 0.27, spot B 0.39, spot C 0.83 and spot D 0.97 all the spots were scraped. Anti-microbial activity was evaluated for all the spots.18 Only spot D was showed very good antibacterial activity at different concentration of 20, 50, 100, 200, 400, 800 and 1200 μg/ml against Brucella spp. The MIC observed was B. abortus (1.5 mm) at 800 μg/mL, B. melitensis (1.7 mm) at 800 μg/mL and B. suis (1.7 mm) at 800 μg/mL concentration. The MIC of Brucella spp. was compared to the standard GN –1.4 mm 1.6 mm, 1.2 mm respectively (Figure 2). Statistical significance was observed for Brucella spp. P≤ 0.05 for the B. suis, P≤ 0.05 for B. melitensis and P≤ 0.01 for B. abortus. Spot D showing good inhibitory activity was subjected to GC-MS analysis and also screened for antibacterial activity against other human pathogens (Table 2).
GC-MS analysis was carried out for the TLC separated spot D (Figure 3) of the ethanolic extract of C. citrinus. Bioactive compounds were characterized and tabulated (Table 3). The total ion chromatograph (TIC) showing the peaks and identity of the compounds is given in Figure 4. Chromatographs of the individual identified compounds are provided in the supplementary file.
Currently, the treatment of brucellosis remains a major public health concern, especially in developing countries.19 In order to increase the treatment efficacy and avoid disease relapse, a classic combination of synthetic tetracycline and aminoglycoside antibiotics has been used. But due to the microbial resistance, multiple drug resistant strains of Brucella have developed. Unfortunately, bacteria have the ability to transmit and acquire resistance to drugs.20 Plants produce secondary metabolites in order to protect themselves from microorganism, herbivores and insects. Even though antimicrobial activities of various medicinal plants have been discovered, very little target compounds have been characterized for activity against Brucella spp.21
The natural plant sources were evaluated to explore antibacterial compounds against Gram negative bacteria B. abortus, B. melitensis and B. suis which are found to be highly pathogenic to human beings. The result of this study showed that the ethanolic extract of C. citrinus exhibited excellent antimicrobial activity against the tested organism including Gram positive and Gram negative bacteria, which is comparable to standard antibiotic effect in table (Table 2). The result of antibacterial activity are in the agreement with the findings of Seyydnejad et al.22 and salem et al.23 but there is no clear evidence of bioactive compounds has been explored for this plant till now. This literature gap prompted us to carry out characterization and test the bioactive compounds against Brucella spp. The result revealed that ethanolic extracts of C. citrinus has promising antibacterial activity.
The C. citrinus extracts exhibited potent antimicrobial activity. TLC is a widely used technique for separation of natural substances and possesses applications in analyzing biologically important compounds, identification and characterization.24 The retention factor values for the plants extracted with ethanol ranged from Rf value of 0.27, 0.39, 0.83 to 0.97. The Rf value 0.97 spot showed antibrucellosis activity and antibacterial activity against other human pathogens. This indicates presence of bioactive metabolites were concentrated in spot D. The individual compounds were screened for the antimicrobial activity but the activity was not observed matching the results of Minqing et al.25 This might be due to the separation of the constituents, which were showing activity at the synergistic level. The antibacterial activity showed by the TLC purified ethanolic extract of C. citrinus could be attributed to the presence of bioactive metabolites. The overall result of the study can be considered as very promising in perspective of new drug discovery from the unknown rare ethnomedicinal plant source, especially because of their medical importance against both bovine and human brucellosis.
Different medicinal plants from the Western Ghats were screened for antibrucellosis activity. Based on the result of this study it can be said that C. citrinus is an effective antimicrobial plant that can be used in biomedical, pharmaceutical field and will be a good source for finding new antimicrobial agents in order to treat and control infections. For the first time we are reporting the antibrucellosis activity in plants Acacia nelotica, T. arjuna, E. jambolana and C. citrinus. C. citrinus showed strong antibrucellosis activity. The bioactive metabolites identified by GC MS were found to have strong antibacterial activity against human pathogens. More studies concerning about the molecular basis of this interaction is important. In future C. citrinus can be assigned as the source of antimicrobial compounds for the treatment caused by the human pathogens.
Brucellosis is an infectious disease caused by gram-negative bacteria Brucella species B. abortus, Brucella melitensis, B. suis, and B. canis. For management, a combination of antibiotics that penetrate the macrophage should be used.
However, because of high rates of treatment failure or relapses due to emerging resistance, the natural resources drugs have fewer side effects than chemical drugs are used as natural therapy.
In the present study different medicinal plants have been screened for antibrucellosis activity and characterized the bioactive component.
The results indicated that ethanolic extract of Callistemon citrinus, showed potent antibrucellosis activity and further identification of its bioactive component against antibrucellosis candidate was warranted for future.