Analysis Of Chemical Constituents Of Pink Lotus Essential Oiland Its Hydrogel Promoting Wound

Objective: Pink Lotus essential oil（PLEO）is a group of volatile components extracted from the flowers of pink lotus.Pink lotus has been used as traditional Chinese medicine since ancient times,but there are few relevant studies on PLEO.There requires the protection of dosage form when used for special purposes due to its volatile instability.In this paper,the chemical constituents of PLEO were analyzed.The antibacterial,anti-inflammatory and cell proliferation promoting activities in vitro were detected.PLEO was prepared into microcapsules,which was mixed into thermosensitive hydrogel materials to prepare hydrogels.And their therapeutic effects on wound healing were explored.Methods: The chemical components of PLEO were analyzed by gas chromatography-mass spectrometry,and its antibacterial activity against Staphylococcus aureus,Pseudomonas aeruginosa and Staphylococcus epidermidis was determined by antibacterial circle method.MTT assay was used to detect cell viability.The inflammatory factor tumor necrosis factor（TNF-α）,Interleukin-6（IL-6）and interleukin-1-β（IL-1β）in human umbilical vein endothelial cells（HUVEC）and human skin fibroblasts（HSF）induced by lipopolysaccharide（LPS）was determined by enzyme-linked immunosorbent assay（ELISA）.Cell migration ability was measured by cell scratch test.β-cyclodextrin was employed to encapsulate PLEO into microcapsules.The process conditions of core-shell ratio,temperature,stirring speed and stirring time were selected by single factor method.The optimal process for the preparation of PLEO microcapsules was determined by simulating the experimental design by response surface method with the encapsulation efficiency as the response value.The morphological characteristics of PLEO microcapsules were observed by scanning electron microscope,and the particle size,potential and polydispersity index of PLEO microcapsules were measured by Malvin particle size meter.PLEO microcapsules were added to the thermosensitive material poloxamer-407 and poloxamer-188 to prepare PLEO microcapsule thermosensitive hydrogels.The morphological characteristics were observed by scanning electron microscope,the swelling degree and p H were measured,and the rheological characteristics were measured by rheometer.A mouse skin incision model was established and treated with PLEO microcapsule thermosensitive hydrogel to observe its effect on wound healing.Hematoxylin eosin staining（HE）and Masson trichrome staining were used to observe the pathological damage and fiber deposition of the skin at the wound.Immunohistochemical staining was used to observe the expression of α-smooth muscle actin（α-SMA）,type I collagen and vascular endothelial growth factor（VEGF）in the skin at the wound.The expression levels of phosphatidylinositol 3-kinase（PI3K）,protein kinase B（Akt）,phosphorylated Akt（p-Akt）,endothelial nitric oxide synthase（e NOS）and phosphorylated e NOS（p-e NOS）were analyzed by Western blot.Results: PLEO was mainly composed of fatty aldehydes（26.57 %）,terpenes（25.64 %）,lipids（14.89 %）and aromatic hydrocarbons（14.2 3%）.PLEO inhibited Staphylococcus aureus,Pseudomonas aeruginosa and Staphylococcus epidermidis,which was significantly different from the blank control group（P < 0.05 or 0.01）.PLEO significantly increased the cell viability of HUVEC and HSF（P < 0.05 or 0.01,compared with the control group）.PLEO can inhibit the production of TNF-α,IL-6and IL-1β in HUVEC and HSF cells induced by LPS（P < 0.05,compared with LPS group）exhibiting anti-inflammatory activity.PLEO significantly promoted the migration of HUVEC and HSF cells（P < 0.05,compared with the control group）.The optimal preparation process of PLEO microcapsules is as follows: core-shell ratio was1:5,temperature was 55 ℃,stirring speed was 550 rpm,stirring time was 3h,and the encapsulation efficiency can reach 71.29 ± 0.019 %.Scanning electron microscope showed that the PLEO microcapsules were spherical,the particle size was 57.03 ± 4.18μm,the zeta potential was about-17.05 ± 1.85 m V and the PDI was 0.338 ± 0.02.The PLEO microcapsules were mixed into thermosensitive materials to prepare hydrogels.The gelation temperature was 36 ℃.Scanning electron microscopy showed that the three-dimensional network structure was good,swelling degree was high,moisture retention was good,and the p H value was 6.48 ± 0.18,which accords with the acceptable range of human skin.Rheological characteristics data showed that the physicochemical properties of PLEO containing microcapsules were similar or even better than those of blank hydrogels.The wound healing rate was significantly increased after PLEO microcapsule hydrogels treatment in mice（P < 0.01,compared with the control group,）.HE staining showed that PLEO microcapsule hydrogels promoted the growth of skin tissue and the morphology of wound tissue was good.Masson staining showed that PLEO microcapsule hydrogels inhibited the inflammatory factors at the wound and promoted the deposition of collagen fibers.In the skin tissue at the wound,the expression of α-SMA,type I collagen and VEGF increased significantly,and the levels of PI3 K,p-Akt and p-e NOS were up-regulated in varying degrees（P < 0.05 or 0.01,compared with the control group）.Conclusion: The composition of PLEO is complex and the content of terpenoids is high.PLEO has the effect of bacteriostasis,anti-inflammation and promoting cell proliferation and migration in vitro.The prepared PLEO microcapsule hydrogels can significantly promote skin wound healing,which may be related to the activation of fibroblasts and the formation of collagen and angiogenesis by activating the PI3K/Akt/e NOS signaling pathway.The experimental results provide a new strategy for the treatment of wound healing and the development and application of PLEO.