Study on the Mechanism of Application of Eucalyptus Volatile oil in Prevention and Treatment of Burn Wound Infections in Vitro

Objective To investigate working mechanism of Eucalyptus volatile oil on the prevention and treatment of burn wound infections. Methods P.a biolm model was used to investigate the effect of Eucalyptus volatile oil on bacterial biolm. The expression of LasI mRNA in P.a was detected by RT-PCR. Results MIC test showed that the volatile oil of Eucalyptus Urophylla in a concentration of 20% or more could exert anti-bacterial effect. However, no zone of inhibition could be observed in the neither high nor low concentration of the Eucalyptus volatile oil. Scanning electron microscopy results showed a signicant delay in volatile oil groups when compared with the control group. The expression of LasI mRNA in the volatile oil group was signicantly lower than that in the control group. Conclusion As Chinese medicine, the volatile oil of Eucalyptus Urophylla can affect the proliferation of P.a and biolm formation by interfering with the expression of LasI, thus achieving the purpose of preventing and treating infection of burn patients.


Introduction
Infection is one of the most common clinical complications of burn patients. It can cause septic shock and multiple organ dysfunction syndrome (MODS), which lead to death [1,2]. Sepsis is a complex process and clinical manifestation which happened in a severely infected person with systemic in ammatory response syndrome (SIRS) [3]. When the blood circulation system is invaded by pathogens, acute systemic infections were caused following pathogens multiplying and toxin production. Endotoxin produced by pathogenic bacteria activates in ammatory cells to produce a large number of in ammatory mediators, which can induce immune dysfunction. It will inhibit the function of immune cells, and then cause septic shock, multiple organ failure, and even death.
Pseudomonas aerugionsa (P.a), is a common pathogen of burn wounds [4], and the most important survival strategy of P.a is the formation of bio lm. In the mature P.a. bio lm, the extracellular polysaccharide ber or the polysaccharide-protein complex is entangled and interlaced into a network, and the gap is lled with a negatively charged alginate, which constitutes the external environment of BF.
And this strong diffuse barrier hinders the penetration of antibiotics, especially positively charged ones [5,6], greatly improving the viability of bacteria [7]. The current studies found that P.a can use Quorum Sensing (QS) systems to regulate toxic factor formation named Las system (lasI/lasR) [8]. LasI encode the signal molecule 3-oxo-dodecanoyl homoserine lactone (3-OXO-C12-homoserinelactone, 3-O-C12-HSL) and transcriptional activator regulatory signaling molecules. Studies have shown that LasR and rhlR gene defects can affect the ability of P.a. to form bio lm in vitro, and that QS system plays an important role in the establishment and chronic development of P.a. lung infection [9,10].
Traditional Chinese medicine has unique advantages in preventing infection caused by bacterial bio lm, but also can inhibit the formation of bacterial bio lm. It has become the focus of further anti-infective drugs following antibiotics base on its wide source of drugs, low price and small side effects [11].
Eucalyptus Urophylla is a kind of Myrtaeeae plant cultivated in Guangxi provinces, The use of Eucalyptus leaves has been circulating in the public. They are applied to the skin to prevent mosquito bites. Modern medicine has con rmed that the eucalyptus leaves contain gallic acid, phenols, mellow alcohol, and eucalyptus.The tests of gallic acid in vitro extracted from Eucalyptus Urophylla leaves inhibited Staphylococcus aureus, Pseudomonas aeruginosa, pneumococcus, typhoid and paratyphoid bacillus, which proved that eucalyptus leaves have good antibacterial and anti-in ammatory effects [12,13]. Therefore, this study aimed to investigate the effect of Eucalyptus urophylla volatile oil on burn patients for prevention and treatment of concurrent infections.

Analysis methods
The Eucalyptus leaf oil was taken and analyzed by GC-MS. The mass spectrum was searched in NIST98 standard mass spectrum database (HPMSD Chem-Station) to con rm each chromatographic peak.

MIC test
Because the volatile oil of Eucalyptus leaves is insoluble in water, the co-solvent Tween-80 is used for co-solubilization, and the ratio of volatile oil and co-solvent is 5: 1000. Take 10 μL 0.5 MCF of PAO1 bacterial suspension and inoculate it into 1 mL of LB medium, and then add soluble volatile oil to con gure a gradient mixed suspension with drug concentration of 10% to 50%, and incubate in a CO2 incubator for 24 hours, and then transfer to the blood plate to observe the growth of bacteria.

protein ngerprint analysis
Mass spectrometer (Micyo ex LT/SH, BD, US) was used for testing and FlexAnalysis (Bruck, US) for analysis. Standard MBT method, usual for typical laboratory standard samples, used for validation and speci cation issues. MS/Parent Mode: On. Initial Laser Power: 30%, and maximal laser power: 40%. Allow Only: 80 satisfactory shots per raster spot. Matrix Blaster: Fire initially 10 shots with a laser power of 40%. [10][11][12] 1.5.1 Bio lm model construction: Take 20mL of LB medium into a 50mL centrifuge tube, inoculate 1mL of 0.5 McFarley bacterial suspension, put a sterile gastric tube(1cm) into the bacterial suspension, and place it in a shaker at an angle of 45 degrees. After shaking for 24 hours, after that a bacterial lm was formed on the surface of the sterile gastric tube, which is a bio lm model. 1.5.2 Preparation before SEM observation: 1) Biological specimens were xed with 3% glutaraldehyde for 2 hours, 2) 0.1 mol/L PBS buffer solution was immersed and washed 3 times for 10 min each, 3)

BF Construction and SEM observation
Osmium tetroxide was xed for 1 hour, 4) 0. 1mol/L PBS buffer solution for 3 times and 10min each time, 5) Ethanol from 50%, 70%, 80%, 90%, 100% (three times) soak and dehydrate for 10min each time, 6) 100% pure six Methyldisilazane was immersed three times for ten minutes each time, put into a vacuum dryer and vacuum dried, 7) Paste the sample to the sample holder, put it on the IB3 ion sputtering instrument and observe it under an electron microscope.

RT-PCR detection
After constructing the bio lm model according to the above steps, the gastric tube was gently washed with sterile PBS and the bacteria on the inner and outer surfaces of the gastric tube were collected with a spatula. After enrichment, the sample RNA was extracted using the TAKARA genome extraction kit and reversed. After 15 minutes of recording, use RT-PCR detector (Redstone SLAN-96P, Shanghai, China) to test the LasI expression.

Statistical methods
SPSS 19.0 and GraphPad were used for statistical analysis and picture processing. Measurement data were analyzed by t test. Sample means were expressed in the form of mean ± standard deviation. The test level α = 0.05. When the P value is less than 0.05, it means The difference was statistically signi cant.

Major chemical components of eucalyptus volatile oil analyzed by GC-MS
The GC-MS test was performed and a total ion current map of Eucalyptus volatile oil was obtained (Fig  1).  Fig. 3a. It was found that the mass-to-nucleus (m/z) was signi cantly different at 7594 and some other places. The differences lead us to consider that Eucalyptus volatile oil has an effect on various proteins of the bacteria and at the same time it interferes the P.a. QS system as well. And we will continue to explore which kinds of these protein are in the future study. But what can be con rmed is most of these proteins are related to bacterial resistance.
As shown in Figure 3b, after the successful construction of the bacterial bio lm model, the surface bio lm morphology was observed by SEM. We found that the control group had formed an early bio lm with a thick state. In the observer group treated with Eucalyptus volatile oil, the bio lm morphology is relatively thin, which can be indicated that the Eucalyptus volatile oil of has inhibitory effect on bacteria, and the formation of bio lm was also delayed.
As shown in Figure 3c, the results of RT-PCR detection of LasI mRNA expression of LasI mRNA in the experimental group was (30.68 ± 0.087) after conversion by standard RT-PCR curve [Y = -3.346LOG (X) +34.52, R = 0.998], and the average value of the control group was (31.12 ± 0.063). LasI mRNA expression in the experimental group was signi cantly reduced after intervention of Eucalyptus volatile oil, and the difference was statistically signi cant (P <0.05).

Discussion
Guangxi Provence is an area lived Zhuang people, with abundant medical resources and culture. Therefore, the research on Chinese medicine is the advantage and characteristic of this article. At the same time, due to the large number of ethnic minorities in Guangxi, its diverse ethnic culture and unique local customs and activities are rare intangible cultural heritages in China, but it also brings problems: reworks and recrackers every year cause a large number of people suffering from injuries, burns, etc., which consume huge medical resources in the treatment of such patients. Therefore, while protecting China's intangible cultural heritage, it is particularly important and necessary to treat the burns as well.
The bio lm-forming system can cause high-resistance of P.a., which is one of the important factors causing clinical intractable infection [14]. Therefore, people are beginning to explore other ways to treat P.a. infection, such as the development of new antibacterial drugs, alginate monoclonal antibody, gene regulation and improvement of biomedical materials, but these methods still cost too much and have a narrow application range, causing clinical application limited [15]. The Quorum Sensing (QS) system plays an important role in P.a. bio lm formation [16], and it is a mechanism of information transmission between bacteria. Cells do not sense the presence of bacteria themselves, but rather the concentration of signal molecules, and the concentration of signal molecules depends on the density of the cells. In the case of the QS system involved in the regulation of the expression of virulence factors, interference with the QS signaling system may affect the regulation of the pathogenic factors of P.a.
The results in this study showed that the Eucalyptus urophylla volatile oil has a lower minimum inhibitory concentration(MIC) and better bactericidal effect, 20% concentration can fully inhibit the growth of Pa, and at the same time, results under scanning electron microscope(SEM) show that after the intervention of P.a. in the Eucalyptus urophylla volatile oil, the formation of bacterial bio lm can be delayed. Many reports have put that the formation of bio lm is closely related to bacterial resistance. This indicates that one of the mechanisms of the Eucalyptus urophylla volatile oil can be used in clinical bacterial treatment is to destroy its drug resistance. In addition, the results of RT-PCR detection show that the QS system related gene LasI mRNA can be reduced by the Eucalyptus urophylla volatile oil, and some studies have found that the secretion, virulence factors (elastin, exotoxin A, pyocyanin, etc.) and bio lm formation of P.a. are regulated by the QS system [17], so we can consider that the Eucalyptus urophylla volatile oil has a signi cant effect not only on bacteriostatic, but also on the virulence-related system.
In summary, the Eucalyptus urophylla volatile oil has high application value in the treatment of clinical infections. Its mechanism of action is mainly manifested in the following aspects: directly inhibiting bacterial growth and reproduction, delaying the formation of bacterial bio lm to destroy its drug resistance, and reducing LasI gene expression so as to reduce its virulence. However, the Eucalyptus urophylla volatile oil also has its shortcomings. For example, the pungent odor is emitted during the preparation process, and its extraction requires professional distillation and condensation recovery equipment. In daily life and clinical departments can not be used on demand.
In the recent worldwide battle against the new coronavirus (2019-nCoV), Chinese medicine has played an important role in patient treatment, rehabilitation, and daily infection prevention [18,19]. Treatment of integrative medicine has made signi cant clinical e cacy in Wuhan, Hubei Province and even the whole China [20]. Therefore, we hope to extend the theory of traditional Chinese medicine to the worldwide medical eld, and in the future research, we will further work on its therapeutic mechanism, but also improve the shortcomings in exploring and promoting its use value.

Declarations
All experiments were performed in accordance with the Federation of European Laboratory Animal Science Association guidelines, and the protocols were approved by the Animal Ethics Committee of The First A liated Hospital of Guangxi University of Chinese Medicine (Nanning, China).

Competing interests
There is no competing interests and all authors agree with the author list.

Author contribution
Lei Yang conceived and designed the study; Leiyang, Xingxin Gao and Qing Huang wrote the manuscript; Zhimin Lin and Songlin Chen collected the data; Xiaodong Huang and Jinpeng Feng analyzed and interpreted the data; Lili Zhao, Yinghui Lin and Guangzong Hua provided critical revisions; Sheng Li approved the nal version of the manuscript.    Figure 3b Scanning electron microscopy (SEM) observation. We found that the control group had formed an early bio lm with a thick state. In the observer group treated with Eucalyptus volatile oil, the bio lm morphology is relatively thin, which can be indicated that the Eucalyptus volatile oil has inhibitory effect on the formation of bio lm.