Evaluation of Antimicrobial Activity of Commiphora myrrh against Standard Bacterial Strains and Clinical Isolates with Chemical Analysis Profiling

Aims: The objective of present study was to investigate the chemical analysis biological activates of commiphora myrrh the chemical analysis of myrrh was analyzed several ways. Study Design: Where wet digestion was used to estimate the concentrations of a number of chemical elements in it, which are of great importance to humans and are also attributed to many of its medicinal uses. Place and Duration of Study: Clinical isolates from wound infections obtained from laboratory of Marjan Hospital Hilla city Iraq during period Fed. 2020 which include (four E. coli and 4 of S. aureus, and 4 pseudomonas aeruginosa ). All clinical isolates were classified and by laboratory of marjan hospital: Hilla city Iraq. Methodology: In order to know the nature of the groups present in it, in addition to the quality of the organic materials, FTIR analysis and G.C analysis were used by using the ethanolic extract, where some of the organic materials within their compositions in identified. Results: The antimicrobial potential of ethanolic extracts of myrrh were studied against many standard strains of gram positive and gram negative bacteria and (12) clinical isolated from patients with wound infections obtained from the bacteriology section of the clinical microbiology laboratory of marjan hospital Hill city/Iraq during period Feb. 2020 to Nov. 2020. The clinical Original Research Article Alasady et al.; JPRI, 33(47A): 714-723, 2021; Article no.JPRI.75566 715 isolates include (4) isolates of staphylococcus aureus, (4) isolates of E.coli and (4) isolates of pseudomonas aeruginosa, and it was confirmed using the usual methods of diagnosis. The broth dilution method was used for determination of the MICs of (minimal inhibitory concentration) of myrrh extract against pathogens under study. Six concentration (80, 60, 30, 12, 6, 3 mg/mL) of myrrh extracts were tested. Conclusion: The result revealed that the highest activity was against S. Aureus at concentration (80, 60, 30 mg/mL) which showed completely inhibition of the growth (100%). While the gramnegative bacteria E.coli and P. auroginosa the concentration (80 – 60 mg/mL) showed 100% inhibition in contrast the concentration (12, 6, 3 mg/mL) showed no activity of myrrh extract against all pathogens under study. The result indicates that myrrh is an antibacterial agent that can be used in the future by making appropriate doses


INTRODUCTION
Myrrh is one of the important and widely used medicinal plants, as it was used by the ancient Egyptians as medicine [1]. It has an economically and culturally, valuable product obtain from commiphora myrrh tree. The natural plant product used in several pharmaceuticals industries ,cosmetics and others .there is many local applications in medicinal ,hygienic and insecticide [2]. It is widely used in traditional medicines for treatments of wide variety of infectious disease. In more recent times, myrrh has been used as a medical antiseptic, as it was used in the mouth when there are cases of ulcers and infections [3]. Scientific classification of myrrh. It belong to kingdom: Plantae ,division : magnoliophyte class : magnoliopsida :order: Spindale's ,family burseraceous :genus commphorra [4]. It is also a small tree with a short true myrrh plant produced by C. Myrrha. Different types of trees are also found in many places such as southern Arabia and northeastern Africa .Myrrh is reddish brown resinous material [5]. The raw myrrh resin and its essential oil upon combustion produce bitter smoke and phenol. Myrrh can be used as antimicrobial ,antifungal and somewhat antiviral ,immune stimulant ,bitter ,circulatory stimulant ,anti-inflammatory and antispasmodic [6] myrrh a popular traditional natural medicine [7]. As for the chemical composition of myrrh from a basic water-soluble gum and an alcohol-soluble resin [8]. Among the uses of myrrh is as an analgesic and is also used to clean wounds, as it was used for this purpose for more than 2000 years until the discovery of morphine [9].
Myrrh is one of the ancient and important medicines because of its use by the ancient Egyptians on a large scale. Recent studies also showed that the extracts of the myrrh plant have great efficacy against various types of pathogens, and many of the compounds that were isolated from this plant were found to have antibacterial activity and proven It is through strong inhibition with minimum inhibitory concentration (MIC) values ranging from 0.2-2.8 Mg/ml [10,11]. Some isolated compounded like sesquiterpenoids inhibited the different pathogens growth with (MIC) ranging between (4-256 Mg/ml) . The aim of the present studs was to evaluate the effect of ethanolic extracts of C.Myrrh as antibacterial against gram positive and gram negative pathologies isolated from patients. it tends not to dissolve very well in water [12] Myrrh contains a 2-8% volatile oil (myrrhol), 23-40% resin (myrrhin), 40-60% gum, and a bitter principle10-25% [13]. The gum contains polysaccharides and proteins, while the volatile oils consist of steroids, sterols and terpenes. Furanosesquiterpenes are also derived from the distinctive aroma of myrrh [14]. The main Chemical compounds of Myrrh [15].

 Extraction
Myrrh was obtained from spice shop (saudia Arabic)

 Evaluate the Antimicrobial Activity
The evaluation of antimicrobial activity of myrrh was done against many standard strains of gram positive and gram negative bacteria and clinical isolates from wound infections obtained from laboratory of marjan hospital Hilla city Iraq during period Fed. 2020 which include four E.coli ,4.s.aureus and 4 psendomonas aurogenosa. All clinical isolates were classified and by laboratory of marjan hospital :Hilla city Iraq . All bacterial isolates were subjected to microscopically examination to ensure their purity and morphology and staining properties using gram staining technique.

Preparation Test Organisms
One ml of 24 hrs broth culture of the test bacteria were aseptically distributed on nutrient agar slant then incubated at 37 °C for 24hrs. the bacterial growth was harvested and washed off with sterile normal saline: produce suspension containing 10 10 colony forming unit (CFU)/ml. then stored at 4°C till used [16].

Determination of Antimicrobial Activity
Serial dilution of myrrh extract test was prepared in methanol as dilution medium since it was devoid of any antimicrobial activity.

Determination of MIC
All test strains were subjected susceptibility test by standard agar dilution method according to the current guidelines of the clinical laboratory standard institute. Plates containing concentration of (3 ,6 ,12 , 30 ,60 and 80 mg/ml) in Muller hunt on (MHA) agar were prepared bacterial suspensions was adjusted to McFarland standard 10 ML of (1.5 × 10 6 ) CFU of bacterial suspension spotted on to the Surface of MHA .it was allowed to spread on the agar for 10min ,then incubated at 37 ˚C for 24 and 48 hrs. plate were read against a dark ,nonreflecting back ground. Growth was interpreted positive if confluent. Weak if a haze forms which is difficult to read and number of colonies formed there are ˃10 and as negative if no growth occurred. Test strains were considered resistant if the growth was positive or weak or if more than one colony was observed.  Before doing the test of MIC against E.coli we ensured the isolates identified by culturing on maccokey agar medium, a large red collies were observed as a result of fermentation also used gram stain technique. Gram negative rods were seen [16]. The effect of myrrh concentration on clinical showed similar results E.coli to Stap. aurous at 80 and 60 mg/ml concentration. But the growth of E.coli showed normal at 30mg/ml and 12, 6 ,3 mg/ml concentration Table (4). found that.

Clinical Isolates
The inhibitory effectiveness of myrrh extract against pseudomonas. aeruginosa was determined in Table ( 5). Myrrh extract had inhibitory effect on p. aeruginosa clinical isolates on macconkey agar plates pale and colonies were observed on nutrient agar plates the isolate produced blue-green pigment which diffused in the surrounding medium . The present study showed that the soluble components of myrrh were highly bactericidal against S. Aurous in (80, 60 ,30)mg/ml conc. also the results showed bactericidal effect on clinical isolates of gramnegative bacteria similar activity of myrrh has been reported by many researches [17]. Myrrh extract produced due to containing number of active constituents such as furan type terpenoids which were shown to possess biological activity . It was also noted in the study, that myrrh extract had a high bactericidal activity on types of standard gram positive and gram negative bacteria. The tested isolates caused variety disease. Resistance to many antibiotics quickly develops, so the observation of the bacteriostatic effect of myrrh extract makes it particularly useful. No microbial resistance against myrrh extract has been reported. According to the results of the present study following recommendation showed be done .due to the coast of antibiotics which regarded as a red problem for many people and appearance of severe antibiotic resistance to many drug this emphasizes the emphasized the need for search for anew cheap a safety antimicrobial drug. We suggested for future work study the toxicological and clinical should be carried out on many selected medicinal plants to prove their safety and therapeutic activity for commercial utilization [17].   (13)

Chemical Analytics
1-It is known that chemical elements are fundamentally important in many physiological functions in humans, so an analysis of the myrrh plant was conducted to estimate some of the elements present in it, which have an important role in the human body and its functions.

FT.IR
From FTIR examination ,notice from the figures the bonds of material that appearance of (O-H stretch) bond in the wave number (2400 cm

GC Technique
Using a GC technique, some of the organic compounds found in the myrrh plant were identified, as 27 organic compounds were estimated. Fig. 1 shows the chromatogram of organic constituents taken by the GC. Results indicate of the organic constituents identified in the myrrh (Syed Rizwan et al 2017 , Berkowitz FE .,2015 ., ).

CONCLUSION
The result revealed that the highest activity was against S. Aureus at concentration (80 ,60 ,30 mg/mL) which showed completely inhibition of the growth (100%). While the gram-negative bacteria E.coli and P. auroginosa the concentration (80 -60 mg/mL) showed 100% inhibition in contrast the concentration (12 ,6 ,3 mg/mL) showed no activity of myrrh extract against all pathogens under study. The result indicates that myrrh is an antibacterial agent that can be used in the future by making appropriate doses CONSENT It is not applicable

ETHICAL APPROVAL
It is not applicable