The Prediction And Verification Of Anti-inflammation,analgesic,Anti-blood Stasis And Anti-acne Functions Of Borneol Essential Oil

The indigenous Chinese tree,Cinnamomum camphora chvar.Borneol has been planted extensively in China.Natural crystalline borneol was extraction from branches and leaves by steam distillation to obtain borneol essential oil(BEO).BEO is a relatively new natural product;therefore,it is important to characterize its bioactive functions and potential medicinal applications.Based on the main components of BEO,this article used network pharmacology,molecular docking,and transcriptomics to predict its potential biological activity.Based on the main function of borneol,a series of functions of BEO were explored,such as anti-inflammation,analgesic,anti-blood stasis,anti-acne,etc.To evaluate these functions,network pharmacology was adopted to construct a“multi-component-multi-target-multi-pathway”mode of action,further analyze its material basis and mechanism of action.Lastly,physiological indicators were adopted to verify the prediction results so that to provide theoretical guidance and data support for the development of BEO-based products.There are five main parts to this paper:1.Compositional analysis and safety evaluation of BEO.Qualitative and quantitative analysis by gas chromatography-mass spectrometry(GC-MS)and gas chromatography-flame ionization detector(GC-FID)showed that BEO contains42 compounds,including monoterpenoids,oxymonoterpenoids,sesquiterpenoids,and oxysesquiterpenoids.The main components of BEO are borneol(16.4%),?-caryophyllene(10.7%),camphor(10.6%),limonene(8.2%),?-pinene(7.5%),myrcene(6.2%),and camphene(4.4%).The median lethal dose(LD₅₀)for male mice was 5081 mg/kg(non-toxic)and that for female mice was 2749 mg/kg(low toxicity).If a BEO solution was applied topically,there was no acute,skin,or eye irritation,at BEO concentrations less than 50%.2.Theoretical predictions and validation of BEO anti-inflammatory bioactivity.We constructed a“component-target-signal pathway”for regulation of inflammation by BEO,by network pharmacology and compared the anti-inflammatory effects BEO and borneol,using the xylene-induced mouse inflammation model.The differences in mechanism of action between BEO and borneol were analyzed by transcriptomics,to verify the main targets and signal pathways influenced by BEO's main component(borneol).The differentially expressed genes(DEGs)obtained from transcriptomic analysis were closely associated with the inflammatory disease genes identified by network pharmacology.3.Anti-inflammatory and analgesic effects of BEO.A hydrophilic BEO nano-emulsion was prepared,with good human erythrocyte membrane stabilization,mediated by inhibiting heat-induced hemolysis(IC₅₀=5.29 mg/m L)and hypotonic solution-induced hemolysis(IC₅₀=0.26 mg/m L)in vitro,suggesting that BEO can also stabilize lysosomal membranes.BEO was topically applied to mice auricles;both single and repeated administration significantly reduced xylene-induced auricle swelling(P<0.0001).Expression of inflammatory mediators,including interleukin(IL)-1?,IL-6 and tumor necrosis factor?(TNF-?)in serum and tissue were significantly downregulated(P<0.05),as were the m RNA expression of IL-1?(P<0.05)and TNF-?(P<0.001).BEO also inhibited the release of the inflammatory mediators,TNF-?,IL-1?,and IL-6,in the lipopolysaccharide(LPS)-induced mouse macrophage inflammation model,in a dose-dependent manner(r=-0.9954,-0.9547,-0.9724,respectively).The analgesic effect of BEO was evaluated by the glacial acetic acid-induced mouse writhing-pain model.Continuous topical application of BEO to the abdomen of mice for 6 d,significantly reduced observed writhing in mice(P<0.001)with a strong dose-response relationship(r=0.9306).Concomitantly,the levels of the serum pain-related mediators,prostaglandin E₂(PGE₂)and transient receptor potential melastatin-8(TRPM8)were significantly reduced(P<0.001),the latter in a dose-dependent manner(r=-0.9427).4.Effects of BEO on improving blood circulation and amelioration of blood-stasis.Using human platelets,we compared the inhibition of platelet aggregation by BEO and borneol in vitro;both of them significantly reduced adenosine diphosphate(ADP)-induced human platelet aggregation,at a borneol concentration?0.02 mg/m L(P<0.0001).At the same borneol concentration,inhibition of platelet aggregation by BEO was stronger than by pure borneol(P<0.001),indicating that BEO components other than borneol contribute significantly to the inhibitory effect.We analyzed the mechanism of BEO regulation of thrombosis by network pharmacology,and identified the key targets of BEO(TBXAS1 and NOS3),in the main platelet-activation pathway.The rat blood-stasis model was applied for verification of these findings in vivo,confirming that serum nitric oxide(NO)and thromboxane B₂(TXB₂)levels,which are regulated by TBXAS1 and NOS3,were partially normalized(P<0.05).In addition,BEO significantly reduced whole blood viscosity(P<0.05)and the erythrocyte aggregation index(P<0.05),in a dose-dependent manner(r=-0.8905?-0.9916).Histopathology also revealed that BEO inhibited inflammatory cell infiltration and tissue-structure destruction in the aorta,myocardium,and lungs,caused by blood stasis in mice.5.Anti-inflammatory and antimicrobial effects of BEO against acne.The minimum inhibitory concentrations(MICs)of BEO and borneol against Staphylococcus epidermidis(the main cause of acne),obtained by the two-fold dilution method,were both 0.5 mg/m L.BEO inhibited the growth of S.epidermidis and significantly prolonged the lag phase of growth.Analysis of the effects of BEO and borneol on the maximum growth rate(?_max)and the time to reach?_max,showed that BEO had stronger antibacterial activity,indicating that BEO components other than borneol contribute significantly to the antibacterial activity.Electron microscopic imaging showed that the integrity of the bacterial cell wall had been disrupted by BEO treatment.In addition,cell membrane integrity was disrupted,as shown by extensive leakage of nucleic acid and protein from the cells into the medium,resulting in eventual cell-death.The human keratinocyte(Ha Ca T)cell proliferation and inflammation model demonstrated that BEO inhibits the proliferation of Ha Ca T cells induced by S.epidermidis(P<0.0001),as well as release of TNF-?(P<0.0001)and IL-1?(P<0.05),both in a dose-dependent manner(r=-0.9952,-0.9492,respectively).A rabbit-ear acne model,induced by intradermal injection of S.epidermidis demonstrated that BEO can ameliorate the severity of rabbit-ear acne.Network pharmacology analysis identified the targets of BEO components in the TNF-?and Toll-like receptor signaling pathways,which were then validated in the rabbit-ear acne model.BEO treatment significantly down-regulated the expression of TNF-?,the concentrations of toll-like receptor 2(TLR2),phosphoinositide 3-kinase(PI3K),protein kinase B(AKT),nuclear factor kappa B(NF-?B),TNF-?and IL-1?in ear tissue,and the concentrations of TNF-?and IL-1?in serum(P<0.05).These results suggest that BEO acts against acne through regulation of the TLR2/PI3K-Akt/NF-?B signaling pathway.Results of study have revealed the material basis and mechanism of BEO functions,including anti-inflammation,analgesic,anti-blood stasis,and anti-acne functions so that to be further applied BEO to industry.