Antibrucellosis Activity of Medicinal Plants from Western Ghats and Characterization of Bioactive Metabolites

Background: Brucellosis is one of the most prevalent bacterial zoonosis which is transmitted to humans from animals. As an alternative to conventional antibiotics, medicinal plants are valuable resources for new agents against antibiotic-resistant strains. Objective: To evaluate the antibrucellosis activity of different medicinal plants collected from the Western Ghats against Brucella abortus, Brucella melitensis, Brucella suis. Identification and characterization of the bioactive metabolites of the potent antibrucellosis agent by Thin Layer Chromatography and Gas chromatography mass spectroscopy. Methods: Antibacterial assay was carried for the ethanolic extract of different medicinal plants, the potential and effective medicinal plants extract was subjected for purification by TLC and the bioactive metabolites were characterized by the GC MS analysis. Results: Acacia nelotica, Terminalia arjuna, Eugenia jambolana and Callistemon citrinus showed the antibrucellosis activity comparatively Callistemon citrinus had the strong antibrucellosis activity. Further the crude sample was purified by TLC profiling, compounds with different retention factor were screened for antibrucellosis activity, and the bioactive metabolites were identified by GC-MS analysis. Conclusion: For the first time the different medicinal plants from Western Ghats were screened for the antibrucellosis activity. The crude and TLC purified Callistemon citrinus ethanolic extract exhibited strong antibrucellosis activity. The bioactive compounds identified were reported for the first time and the bioactive metabolites identified exhibited as potential antibacterial agents against brucellosis and other Human pathogens.


INTRODUCTION
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. 1Among 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. 3The 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. 5Brucellae genus shows little variation genetically, presently eleven Brucella species have been recognized, they are genetically very similar although each have different host preferences. 6Brucellae 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. 7nfectious 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. 8,9In 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. 10Furthermore, 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. 11edicinal 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

Extraction procedure
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 10 8 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. 16,17he 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 (R f ).Values were calculated for each spot using the following formula: f distance travelled by the solute from the point of application to the center of spot R distance travelled by the solvent front = Preparative TLC was carried out to isolate the separated compounds based on R f 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 10 8 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. 16,17The Petri dishes were subsequently incubated at 37°C for 48 to 72 h anaerobically and the inhibition zone around each 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. 12Screening 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. 13For 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. 1In 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. 14Moreover, they might produce secondary antimicrobial metabolites as a part of their normal growth and development or in response to stress. 15ence 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.

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.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.

Statistical analysis
All experiment/measurements were made in triplicate, and all the values are expressed as the mean ± SE of three independent replicates.Statistical significances were analyzed using two-tailed Student's t-test and means were compared at the level of p ≤ 0.05.

Antimicrobial assay
The antibrucellosis activity was evaluated for different ethnomedicinal plants using disc diffusion method and represented in table (Table 1)

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 R f 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. 18Only 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 study
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

DISCUSSION
Currently, the treatment of brucellosis remains a major public health concern, especially in developing countries. 19In 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. 20Plants 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. 21he 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. 24The retention factor values for the plants extracted with ethanol ranged from R f value of 0.27, 0.39, 0.83 to 0.97.The R f 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

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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.

CONCLUSION
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.
. 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

Figure 2 :
Figure 2: Zone of Inhibition from TLC purified ethanolic extract of C. citrinus Ethanolic extract of C. citrinus subjected for TLC and bioactive spot was identified and different concentration 25, 50, 100, 200, 400, 800 and 1200 µg/ml was tested against B. abortus, B. melitensis and B. suis.Compared to standard antibiotics GN-Gentamycin.The MIC concentration was found to be 800 µg/mL compared to standard GN(10 µg ).

Figure 4 .S125Figure 3 :
Figure 3: Purification of active spot from ethanolic extract of fusing TLC The ethanolic extract of C. citrinus was subjected for identification and separation of active spot using chloroform-methanol (99:1, v/v) as mobile phase.The retention factor (R f ) value of separated spots was determined by calculating the distance migrated by the solvent between the origin (OR) and solvent front (SF) is indicated.The separated spots were identified by exposing plate under UV lamp at 254 nm and calculated R f value for the ethanolic extract of C. citrinus.

Figure 4 :
Figure 4: GC-MS Chromatogram of TLC purified ethanolic extract of Callistemon citrinus showing the bioactive metabolites.The total ion chromatograph (TIC) showing the peak identities of the compounds identified.

Table 1 :
Screening of ethnomedicinal plants for antibrucellosis activity Different medicinal plants were collected from Western Ghats of Karnataka, India.The ethanolic extracts of different medicinal plants were assessed against different Brucella spp.such as B. abortus, B. melitensis and B. suis."-" indicates No inhibition "+" indicates low Inhibition "++" medium inhibition "+++" indicates strong inhibition.C. citrinus ethanolic extract showed very strong inhibition and against B. abortus, B. melitensis, and B. suis.