Synthesis of Cinnamum zeylanicum and Acacia nilotica Extracts and Their Antibacterial Activity against Staphylococcus aureus and Streptococcus pyogenes

micro-Abstract: Different plants are used medically and thofese therapeutic plants have great importance for healing contagious wounds. This herbal treatment is actually also a substitute of different antibiotics and having less side effects on intestinal systems of animals. The foremost concern of this study was to observe the antibacterial activity of Cinnamum zeylanicum and Acacia nilotica . Pathogenic bacteria obtained from wound samples and later identified by biochemical and molecular characterization. Methanol (an organic solvent) was used to extract Cinnamum zeylanicum and Acacia nilotica to check their antimicrobial exertion by using agar diffusion method. Different antibiotics such as, ampicillin, oflaxocin, ticarcillin and cefexime, showed their susceptibility toward antibiotics. The zone of inhibitions for antibiotic and plant extracts’ antibacterial activity were measured. Pathogenic bacteria were identified as Staphylococcus aureus and Streptococcus pyogenesby molecular characterization. These bacteria showed susceptibility to antibiotics and also the plant extracts. Antibiotic oflaxocin showed maximum activity against these two pathogens (12.25 ± 0.44 and 12.375 ± 0.47) while antibiotic cefixime showed minimum effect (1.25 ± 0.28 and 0.625 ± 0.25). Plant extracts showed significant antibacterial activity with maximum activity (14 ± 0.9 by Acacia nilotica and 12 ± 0.5 by Cinnamum zeylanicum ) in 100% solution. It can be concluded thatmethanolic extract of traditional therapeutic plants proved to be a promising source of antimicrobial agents against antibiotic resistant bacteria. Cinnamum zeylanicum and Acacia nilotica were observed to be competent as antibacterial tool against pathogenic bacterial strains.

organisms 7 . Due to continuous and regular use of same antimicrobial agent, microbes become resistant. Therefore, it is the need of time to develop antimicrobial agents with different and novel sources like plants 8 .
Including different things such as herbal medicines, Cinnamon is the oldest one and its history is also included in different Chinese books of about 4000 years back 9 . Cinnamon has been customarily cultivated in Asian countries and it is long lasting tree. Cinnamon belongs to family Lauraceae. Cinnamon have bark different beneficial properties such as antioxidant, antiulcerogenic, antiallergic and antipyretic. Acacia nilotica is the member of family Mimosaceae and in India it is commonly known as babool and in Pakistan known as kikar . Acacia nilotica forests are found in tropical areas of India and Pakistan and having great value for fuel, fodder, tannin and small timber. These plants are distributed in Australia, America and Asia and their beneficial aspects are confirmed for the cure of gonorrhea, wounds, diarrhea and leucorrhoea 10,11 . The aim of the current research was to study the antibacterial activity of plant extracts against common pathogens especially Staphylococcus aureus and Streptococcus pyogenes. The purpose of this study was to explore biological ways of controlling bacterial infections rather than using antibiotics as bacterial pathogens are becoming resistant to commonly used antibiotics.

Bacterial sampling and isolation of bacteria
Bacterial sampling was done randomly from operation theaters of Jinnah hospital, Lahore during summer period of year 2020. 20 samples were collected from untreated wounds of patients by swabbing with the help of sterilized cotton swabs. Swabbing was done with the help of on duty doctors in hospital with prior permission from hospital authority and consent from patients. Research synopsis was approved by ethical committee/ BOS Board of studies of department of Zoology, GCU, Lahore. Samples were brought to Microbiological laboratory GCU, Lahore, by using saline solution of 0.85 in sterilized sample tubes. In laminar air cabinet, swabs were spread on agar plates with the help of glass spreader. The agar plates were incubated at 37 for a day. Bacterial colonies can be differentiated by various shapes and symmetry after incubation process. The only isolated colony of bacteria was selected to isolate the pure culture and was selected with a sterilization loop and placed on new plate. The antibiotic susceptibility of the bacterial isolates was checked against ampicillin AMP oflaxocin OF , cefexime CFM and ticarcillin TI . The plates were incubated at 37 for 24 hours. Different zones appeared after incubation depending on the sensitivity of the bacterial strains 13 . Inhibition zones were established around the bacterial strains which showed sensitivity towards relevant antibiotic Fig. 1 .

Collection of plant materials and solvent extraction
From Botanical Garden of Government College University, Lahore, leaves and branches of A. nilotica were collected while Cinnamon bark was purchased from market. Leaves, branches of plants were thoroughly washed under tap water and once with sterile distilled water. After that, all parts of plant used in study were dried in shade of the wind. After drying, 25 g A. nilotica in fine powdered form dissolved in 100 ml of methanol 1:4 and this solution was incubated for 1-7 days. After 7 days filtration and evaporation of the solution was done and extracts were dried in oven at 60 . The concentrated extracts were finally soaked in methanol in the ratio 1:6 14 . Although methanol is considered a toxic solvent, but it is extremely useful for the extraction of certain phytochemicals due to its polarity. It is safe to use as it is highly volatile and is completely evaporated from extracts. As methanol is completely evap- orated, so the antibacterial activity is done only by the plant extract.

Antibacterial activity of plant extracts
With the use of Kirby-Bauer disc diffusion method, antimicrobial activity of plant extract was checked. The paper discs were made of a sterile filter paper and soaked in plant extracts of different concentrations. Sterile filter paper discs were placed on the agar plate by using sterile forceps. For a control group, 100 methanol was used and plates were incubated at 37 for 24 hours 15 . By using millimeter scale, diameters of inhibition zones were measured Figs. 2 and 3 .

16S rRNA gene sequencing of bacterial strains
Phenol chloroform extraction method was used to isolate genomic DNA of bacterial isolates Fig. 4 . Afterwards, amplification was done using universal primers 16S-27F 5 -AGAGTTTGATCCTGGCTCAG-3 and 16S-1522R 5 -AAGGAGGTGATCCAGCCGCA-3 in a Techne thermal cycler Progene . Polymerase chain reaction was done, under most favorable conditions, as shown in Table 1. The reaction mixture was prepared by mixing the following chemicals in measured quantities Fermantas in sterilized PCR tubes Fig. 5 . The genes of PCR product was purified by using GF-1 DNA recovery kit by Vivantis. After then, for

Results
In current study, 25 samples of post-operative wounds of patients taken from Jinnah Hospital, Lahore, were used. Out of these 25 samples 10 strains were separated out in order to perform more biochemical tests to check their antimicrobial susceptibility. Gram positive coccus GC1 was found in the form of clusters, as a result of gram staining, GC1 at genus level after executing different biochemical and artificial media tests was identified as Staphylococcus species which was non-mobile in moving medium. β haemolysis was shown by GC1 on blood agar medium. This strain was identified at molecular level by ribotyping Fig.  6 . Gram positive coccusGC2 was visible in the form of chains as a result of gram staining. After incubation, the strain was observed non-motile in motility agar. After carried out various biochemical and microbial tests, GC2 was recognized as Streptococcus species. β haemolysis was shown by GC2 on blood agar medium. The bacteria was identified by molecular characterization Fig. 7 . Antibiotic resistant strains were also subjected to the plant extract having antimicrobial qualities and check their sensitivity towards antibiotic extract of plant. The antibiotic susceptibility of bacterial strains is shown in Table 2. Bacterial strains showed sensitivity against Cinnamum zeylanicum and Acacia nilotica as shown in Table 3. Both of these strains showed minimum to maximum sensitivity to antibiotics and to the plant extracts. Antibiotic ofloxacin showed maximum antibacterial effect against these two pathogens

Discussion
The development of antibiotic resistance has been recognized since the early stages of antibiotic treatment have expanded to some extent in recent years. This resistance can lead to increased mortality and the cost of health care 16 . In current study, antibiotic resistant strains were selected and then tested for the antibacterial effect of medicinal plants. The bacterial pathogens were identified by biochemical and molecular characterization. Using the technique of amplification of 16s rRNA gene, the sequences were compared with deposited sequences in NCBI for identification of these pathogenic bacteria. Ribotyping has become very effective technique for identication of unknown bacteria 17 . With the passage of time, microbial growth in wound increase and microbes modified themselves which causes more pain and patient feels more discomfort and then it requires more time for healing of wound. Apart from streptococcus, the most common pathogens of wounds are Staphylococcus aureus. Disease caused by infected wound is multifactorial and consists of wireless factors of local and systemic host immune mechanisms of microbes. Most of the studies showed that the major reasons of microbe s resistance towards antimicrobial drugs: overuse and misuse of antimicrobial agents and its contamination, improvement in pathogens and trans-mission of multi-resistant microbes among humans. Because of antimicrobial resistance in microbes, there is an increase in death rate and cost of health care treatment. It has recently been described as a threat to global power and national security. The development of antibiotic resistance has been recognized from an early age, although antibiotic therapy has increased in the past few years 18 .
Mostly, all serious infections and wounds are contaminated by microbes, but it s also a fact that many microbes are beneficial for healing wounds. Most of the contaminants which are beneficent for healing wounds come out from local microflora of infected area. Staphylococcus aureus, Beta-hemolytic Streptococcus S. pyogenes, S. agalactiae , Klebsiella, anaerobes, Proteus, Acinetobacter, E. coli, and Pseudomonas are usually present in an infected wound. Mostly people in under-developed countries are preferred herbal treatment and use of conventional drugs due to their low income. As, conventional healers claims that their drugs, medicines are available at reasonable price and having little or no side effect in contrast to unnaturally prepared medicines. In previous ten years a study was carried out in order to test the antimicrobial effect of Cinnamum zeylanicum against multi drug resistant strains MDR and these strains were found to be sensitive against plant extract 19 . Goni and his coworkers in 2009 carried out a study with a combination of clove essential oil and cinnamon essential oil against the growth of Staphylococcus aureus Gram positive bacteria and E. coli, Pseudomonas aeruginosa Gram negative bacteria 20 .
Arshad and his coworkers in 2017during their work with the methanolic extract of Acacia nilotica found, this extract was influential for all microbes involve in this study. Different microorganism such as P. aeruginosa, S. typhimurium, E. coli and K. pneumoniae showed sensitivity to Acacia nilotica extract 21 . A study was carried out by Deshpandeand Kadamin 2013 in which extraction of Acacia nilotica was done with ethanol and petroleum ether and resulted that both extractions hampered the development of all microbes involved in study. However, microbes showed sensitivity towards ethanol extraction than that of petroleum ether. Theyalsochecked antimicrobial exertion of Acacia nilotica against S. mutans and microbes showed 31 mm inhibition zone with methanol extraction 22 . It is concluded, that Acacia nilotica extract with ethanol have a great inhibitory effect against S. mutans 23,24 .

Conclusions
This study together with earlier reports concluded that the methanolic extract of traditional therapeutic plants proved to be a promising source of antimicrobial agents against antibiotic resistant bacteria. Methanolic plant ex-tracts are safe to use because not only well dissolved in methanol but also become methanol free completely. Methanol is highly volatile, so after thorough process of drying, the extracts become methanol free and safe to use. As the microbial resistance increases due to unnecessary and over prescribed antibiotics, these new sources of antimicrobial agents are effective against human pathogens. Throughout the world studies have found a thousand of phytochemicals and other active components in plants that inhibit growth of pathogenic bacteria. The medicinal plants used in this research are commonly found and proved effective than antibiotics used. Moreover, further research could help identify the nature of active chemical components found in medicinal plants.

Conflict of Interest
There is no conflict of interest among authors.

Author Contributions
NMA designed and supervised the study.HMT carryout the observations and calculations. MKK and KK performed experimental work. BM and KUK wrote first and final draft of manuscript. MC contributed in study design and experimental work. MD contributed in experimental work and data analysis. All authors approved final draft of manuscript.