Antimicrobial activity of plant based Essential Oils and Synergistic effect against selected Human Pathogenic Bacteria

The occurrence of multidrug resistant by pathogens is a universal issue for providing tolerable treatment for various infectious diseases. The predictable anti-microbial proxies are relatively active against plentiful strains, still need of more drugs against multidrug resistant pathogens. Herbal drugs have potential against antimicrobial activity from ancient days and their treatment of pathogenic diseases is increasing for developing plant based natural products. Essential oils have combat against antibiotic resistant bacteria. Thus our study was focused on plant based essential oil against human pathogenic bacteria. The necessary oil extracted from Curcuma longa and C. martini revealed protuberant anti-microbial accomplishments against B. subtilis, Escherichia coli, P. aeruginosa and S. aureus. Low concentration of C. longa oil at 10 μl inhibited the growth of all strains Bacillus subtilis followed by E. coli and S. aureus. Maximum bustle was noted in P. aerogenosa at 40 μl in both C. longa and C. martini oils. Human pathogenic strain treated with C. martini oil showedmaximum inhibition in P. aerogenosa followed by B. subtilis, E. coli and S. aureus. Synergistic activity of oils against the strains showedmaximum inhibition at 40 μl in P. aerogenosa.


INTRODUCTION
Disease caused by infections are increasing day by day in recent years. It is addressed as a second foremost cause of death in the world population (Luqman et al., 2005). Resistance developed by many pathogens is the main reason for inactive antibiotics (Mothana and Lindequist, 2005). This resistance can be caused by many reasons such as unselective use of extensive ield antibiotics, intravenous catheters, organ replacement, etc. (Selvamohan et al., 2012). About 2.22 million hospitalized and one lakhs six thousand patients expired in a solitary year because of adverse drug reactions (Joshi et al., 2011). Due to multidrug resistance and synthetic microbial drugs there is an urgent need to combat the issue by a potential antimicrobial substances from natural origin (Wigmore et al., 2016).
Therapeutic lorae and its foodstuffs are used as traditional medication for eras. As there are many synthetic medicines for diseases, still eighty percentage of the populace in the world depends on ethnomedicine for their therapeutic determinations (Pallant and Steenkamp, 2008). More than nine thousand innate plants have been acknowledged and chronicled for their therapeutic possessions.
Vital oils from therapeutic and aromatic plants are more aromatic in nature because of the availability of secondary metabolites such as aldehydes, terpenes, alcohols, ethers, esters, phenolic and ketones . They remain employed for armothersphy and many ailments such as cardiovascular disease, alzheimer, diabetes, malignancy etc, (Ali et al., 2015). Studies against pediatric patients also reveals no side effects (Man et al., 2019). It has been studied by some researcher regarding impact of essential oil against microbial strains (Sa i and Al-Mariri, 2014).
Curcuma longa Linn is an herbaceous plant of Zingiberaceae family extensively used as Indian traditional remedy for, rheumatism, wound healing, indigestion and several eye ailments (Aggarwal et al., 2007). In India, China and South East Asia it is utilized as a food preservative and coloring agent in various aspects. It also used wide for its biological activities such as anti-in lammatory (Aggarwal and Harikumar, 2009), anti-bacterial activities (Naz et al., 2010).
Cymbopogon martini Roxb. commonly known as palmarosa, which shows more bene icial activity against central nervous system pathologies such as neuralgia, epileptic and anorexia (Buch et al., 2012). It has a potential as antimicrobial, antigenotoxic and antioxidant agent (Sinha et al., 2011;Thaker et al., 2009). Crucial oil from this herbal plants are produced by countries like India, Brazil and Madagascar for different purposes. The aim was to inspect the anti-microbial potential of C. longa and C. martini oils against human pathogenic bacterial strains and their potential on synergistic activity.

Extraction of oil
The healthy fresh leaves of Curcuma longa and Cymbopogon martini were collected during early hours from the ield near Southern Western Ghats nearby Thiruvanaalai (krishnankovil), Virudhunager District, TN, India. The collected leaves of C. longa and C. martini were chopped inely and subjected to hydrodistillation for 4 hours in Clevenger kit. The Cl-EO and Cm-EO separated were placid in a hygienic glass container. Water precipitations were detached using anhydrous Na 2 SO 4 and the Cl-EO, Cm-EO were kept at 4 • C for the auxiliary studies.

Microorganisms
Bacterial strains B. subtilis, E. coli, P. aeruginosa and S. aureus acquired from "Microbial Type Culture Collection and Gene Bank" (MTCC), Chandigarh, India. The cultures were preserved by sub-culturing peri-odically in nutrient agar slants and preserved at 4 • C.

Screening of anti-bacterial activity
Anti-bacterial activity was con irmed by well diffusion method (Mukherjee et al., 1995). The bacterial inoculum was prepared on tryptone soya agar for overnight. The nutrient media on sterile petriplates were kept for solidi ication. About 6mm diameter four wells were done by sterile micropipettes. The test organisms were swabbed into sterile nutrient medium petriplates. Different concentrations such as 10, 20, 30 and 40 ppm of C. longa and C. martini were used for the assay and the inoculated plates were gestated at 37 • C for 24 hours. The diameter of the inhibition zone around each well was measured for anti-bacterial activity. Each investigation was completed in triplicate. The mean diameter of the inhibition zone was documented.

Statistical Investigation
Information from tests were exposed to investigation of and information were communicated as a mean of ive recreates. Critical contrasts between treatment bunches were broke down by Tukey's various range test (noteworthiness at P < 0.05) utilizing Minitab ® 17 programme.

RESULTS
Antimicrobial activity of C. longa and C. martini oils against human pathogenic bacteria including two gram positive (S. aureus, B. subtilis) and two gram negative strains (E. coli, P. aeruginosa) using well diffusion method exposed upright results. The result reveals that both the oils has its potential to effectively suppress the microbial growth of human pathogenic bacteria with variable potency. The activity of oils against pathogenic bacteria showed concentration dependent manner. The oil of C. longa was the virulent oil to retard microbial growth of all tested pathogenic bacteria at higher concentration. Retardation of microbial growth was maximum at 40 µl in P. aerogenosa followed by E. coli, B. subtilis and B. subtilis. Minimal activity was noted in P. aerogenosa at 10 µl followed by S. aureus, B. subtilis and E. coli. Results obtained was shown in Table 1 and Figure 1 .
The activity of C. martini against human pathogenic bacteria reveals the P. aerogenosa was most susceptible than other strains. Most ef icient activity of 42 mm was observed at 40 µl in P. aerogenosa followed by B. subtilis, E. coli and S. aureus. Lower inhibitory activity was observed at lowest concentration of 10 µl in B. subtilis followed by S. aureus, E. coli and P. aerogenosa. Results obtained was shown in Figure 2.   Comparing the results obtained from Cl-EO, Cm-EO oils, the most notable activity was observed in C. longa treated bacterial strains. It showed maximum activity at concentration of 40 µl followed by 30 µl, 20 µl and 10 µl. Synergistic activity potential of Cl-EO, Cm-EO oils showed increased antimicrobial activity with lower concentrations. Results obtained was shown in Table 2 and Figure 3.
The usage of essential oils have been raised day by day and have wide range of claims in diverse areas such as beautifying, medication, therapeutic, agricultural and nutrition industries (Bakkali et al., 2008). Investigation of its biological activities has been one of the attractive research area in which the present researchers are af ianced.
Resistance developed by many pathogens against antibiotics are now become a global threat. Mul-tidrug resistant (MDR) developed by pathogens of many infectious diseases occurs due to repeated usage of most common drugs for their multiple role of action. About ninty to ninty ive percentage of human pathogenic strain S. aureus are impervious to penicillin and seventy to eighty perecentage against methicillin (Casal et al., 2005). In past, a successful antibiotics which are developed should have only the capability to control the infectious disease. But in present days number of factors to be achieved or overcome such as resistance of microbes, persistence level, mode of action, non-development of cross resistance, eco-friendly etc.
Resistance to antimicrobial drug therapy can be acquired by signal transmission within bacterial species (Blair et al., 2015). The resistance mechanism can be occur by transformation in the target  (Abreu et al., 2012). Microorganisms can create obstruction by means of extracellular medication ef lux interceded by ef lux siphons, target change and enzymatic corruption of medications (Ullah et al., 2013).
Essential oils of C. longa and C. martini against human pathogenic bacteria exhibited superior antimicrobial activity. As it is from plant based herbal oil it can be a safest tool for protection. A report from a review reveals herbal based extract exhibited higher antimicrobial activity when compared with synthetic antimicrobial agents (Ayaz et al., 2019).
As our oils showed better results, their action may be breakage of cell membrane, coagulation of organelles, binding and blocking of intake of nutrient sources. It is evident by the report that essential oil involves in commotion of the cell membrane and infuriating cell compound seepage, congealing of cell compounds and interfaces with ATPase membrane proteins, this leads to penetrable of molecules or ions, and inhibit synthesis of enzymes or the nutritional absorption, which cause bacterial death (Cristani et al., 2007).
At 40 µl the essential oils against E. coli was markable in their inhibitory activity.
Synergistic activity of oils showed good inhibitory level when compared with individual oils activity. Thus it has good inhibition ability to control human pathogenic bacteria in minimal level than individual oils. Correspondingly the synergistic effect of two or additional indispensable oils against humanoid pathogens deliberate by numerous scholars also proclaims the same results (Nazzaro et al., 2013).
Combined activity of oils showed effective antimicrobial activity at lower concentrations. This demonstrates the bene icial synergistic potential of C. longa and C. martini oils. Similarly, antibiotics neomycin and amikacin reduction was observed when combined with Lippia origanoides against Staphylococcus aureus (Barreto et al., 2014). In the same way the combination of A. triphylla and L. alba oils against Aeromonas spp reduced effective antimicrobial concentrations (de Souza et al., 2017).
Even though many antibiotics had developed and are presently in markets, there are still requirement of new novel drugs to suppress the microbial pathogens because of drug resistance. The usage of synthetic drugs are limited because of its carcinogenic effects, acute toxicity . In this aspect the natural plant based oils can be used to control epidemic multi drug resilient pathogenic microorganisms (Mulyaningsih et al., 2010).

CONCLUSION
The essential oils from C. longa and C. martini showed signi icant antimicrobial activity, but its inhibitory level increased with lower concentration through synergism. This reduced dosage can contribute decreased bacterial resistance. Thus ef icacy of traditional antibiotics can be governed by our essential oils. Further work has to be focused on synergistic activity of essential oils with conventional antibiotics.

Funding Support
The authors declare that they have no funding support for this study.