Study On The Preparation Technology Of Mucoadhesive Wafer And Effect On Promoting Oral Ulcer Healing Based On The “Combination Of Medicines And Adjuvants” Dual Features Of Bletilla Striata Polysaccharide

Efficient systems for local action can provide a more targeted therapeutic choice,reducing drug dosages and the hazard of systemic adverse and side effects.Therefore,mucoadhesive drug delivery is one of major treatments of oral ulcer.At present,the selection and modification of mucoadhesive excipients has become a hotspot——enhancing the adhesion strength of the drug delivery system,inhibiting the activity of enzyme in the oral cavity to improve the stability of drugs,enhancing the permeation and diffusion of drugs in the oral mucosa,etc.The functionality of bio-adhesive material has been paid more attention by scholars.Bletilla striata polysaccharide?BSP?is a kind of natural high molecular weight material,and it has been widely investigated as a biomaterial with satisfactory mechanical features and unique biological functions.It exhibited superior material properties when used as an antibiotic carrier for topical ophthalmic drug delivery,as a vector for macrophage-targeted gene delivery,and as a wound dressing.As for its pharmacological activity,the polysaccharide also displayed excellent curative effects on wound healing,as well as anti-ulcer,anti-inflammatory,anti-oxidative,immunomodulatory,antifibrosis,and anti-aging activities.From previous research,BSP played important chemical and mechanical roles,indicating that it may be an excellent bio-adhesive material with the potential to treat oral ulcers.Although BSP has been paid enough attention to both material properties and biological activities,few literatures combined the two to highlight the application value of BSP in both aspects simultaneously.Besides,there is little reported in the literature about its effect on oral mucosal lesions.For the aforementioned reasons,a trial has been conducted on BSP?a biocompatible,biodegradable natural polysaccharide with myriad pharmacological activities?,to formulate mucoadhesive wafers and to explore the effect of BSP on the healing of oral ulcer,in order to provide some ideas for the development of drug delivery system for the treatment of oral ulcer.PurposeIn this study,BSP was used as raw material to prepare oral mucosal adhesive wafers,and its effect on the healing of oral ulcer was investigated.The physicochemical properties and structure of BSP were analyzed;The formulation and preparation technology of oral adhesive wafers were optimized,and the pharmacological characteristics of wafers were also characterized;The in vitro anti-inflammation,antioxidant,antimicrobial and in vivo experiments were performed to study the pharmacological activities of BSP.Methods1.The basic physicochemical properties of purified BSP,obtained by our previous extraction method,were investigated by ultraviolet spectrum scanning,phenol sulfuric acid method,Coomassie brilliant blue method,high performance gel permeation chromatography and PMP?3-Methyl-1-Phenyl-5-pyrazolone?-labeling procedure.2.The structure characteristics of BSP were analyzed by using periodic acid oxidation,Smith degradation,methylation combined with gas chromatography?GC?,Fourier-infrared spectroscopy?FT-IR?,Nuclear magnetic resonance?NMR?spectroscopy,gas chromatography-mass spectrometry?GC-MS?,etc.3.A 3² factorial design was employed for optimization of wafers with concentrations of BSP and glycerin as independent variables.Tensile strength,mucoadhesive strength,and swelling index were measured as response variables.Further characterization of the optimal wafer,including folding endurance,surface pH measurement,rheological property,X-ray diffraction?XRD?analysis,scanning electron microscopy?SEM?observation,and thermal analysis,were researched.4.A cytotoxicity assay was analyzed using CCK-8 method to evaluate the biocompa-tibility of systems.The anti-inflammatory activity of the BSP wafer on LPS-activated RAW264.7 cells was also verified.5.The antioxidant activity of BSP was evaluated by DPPH free radical scavenging assay,hydroxyl radical scavenging assay and superoxide anion radical-scavenging assay.6.The in vitro antibacterial activity of BSP wafers against Helicobacter pylori was studied by agar diffusion method.7.The BSP wafers were applied in the acetic acid induced ulcers.The effect of BSP on oral ulcer healing was evaluated by the healing rate of each observation day and histology results.Results1.No absorption peak was detected at 260 nm and 280 nm;The negative responses to Coomassie brilliant blue G-250 evidenced the absence of protein in BSP;The total carbohydrate content of BSP was 63.46%according to the phenol?sulfuric acid method.According to HPGPC analysis,the Mw and Mn of BSP were 3.73×10⁵ g/mol and 6.75×10⁴ g/mol,respectively;By comparing the retention time between monosaccharides in BSP and standards,BSP was a heteropolysaccharide and composed of mannose and glucose,with the molar ratio of 2.946:1.Other sugars,such as rhamnose,galactose and arabinose,were not found in BSP.2.In periodate oxidation assay,the absorbance of polysaccharide at 223 nm was stab-le after 5 days,decreasing at 0.494.The linear equation of the sodium periodate standard curve is:Y=0.0389x+0.0017?R²=0.9995?.According to calculation,1 mol glycosyl consumed 0.95 mol NaIO4 and produced 0.175 mol HCOOH during oxidation.glycerol,erythritol,glucose and mannose were detected in the degradation product;According to GC-MS,the glycosidic bond configuration of BSP mainly consists of Man-?1?,?2?-Glc-?1?,?2?-Glc-?1?,?3?-Glc-?1?,?4?-Man-?1?,?4?-Glc-(1?;The results of FT-IR and NMR indicated that some characteristic chemical groups,which are ubiquitous in polysaccharides,were existed in BSP.And there were a large number of?-1,4-pyranose-related chemical shift signals in the ¹H NMR spectrum and ¹³C NMR spectrum.3.The mathematical relationship between independent variables and responses was analyzed by applying the computer optimization technique.The constant and regression coefficient for Y₁?tensile strength?:Y₁=4.18-0.32X₁+0.057X₂-0.8X₁X₂-0.31X₁²+2.04X₂²;The constant and regression coefficient for Y₂?mucoadhensive strength?:Y₂=1.68-0.01X₁+0.035X₂-0.11X₁X₂-0.46X₁²+0.085X₂²;The constant and regression coefficient for Y₃?swelling index?:Y₃=30.92+0.25X₁-5.31X₂.The tensile strength,mucoadhensive strength and swelling index of the optimized formulation is6.318±0.52 N/mm²,1.797±0.02 N and 24.63±2.53%,respectively.The wafers exhibited sufficient mechanical strength with no breaks or cracks,even after being folded 300 times.Surface pH of the wafers fluctuated between 6.7 and 7.0;Micrographs of the optimized formulation presented a porous interconnecting network;The rheological profiles of the rehydrated wafers and gels before drying presented shear thinning with pseudoplastic flow behavior,and the apparent viscosity is 400.9 mPa·s and 405.23 mPa·s,respectively;The diffraction curve of BSP power contained only a few small peaks and no sharp peaks,manifesting as amorphous structure,while mucoadhesive wafers had a greater intensity compared with BSP power;DSC/TGA thermograms showed the presence of three peaks weight loss events and decomposition patterns.4.The results of cytotoxicity test showed that the viability of RAW264.7 cells were99.14±6.99%,101.64±6.04%,106.48±5.66%,98.79±6.97%,123.34±2.31%and104.63±10.25%respectively in different BSP concentrations?25⁸00?g/mL?.NO,TNF-?and IL-1?were selected to evaluate the in vitro anti-inflammatory activity of BSP wafers.The results confirmed the anti-inflammatory activity of BSP.5.The anti-oxidation activity of the BSP showed that when the concentration of poly-saccharide changed from 0.5 mg/mL to 8 mg/mL,the scavenging rate of DPPH radical increased from 14.25±3.18%to 51.72±2.59%;the scavenging rate of hydroxyl radical increased from 13.10±2.45%to 34.80±2.99%;while the change of superoxide anion radical scavenging rate was relatively steadily,ranging from 21.05±0.91%to 24.15±0.49%.6.The results of antimicrobial activity test showed that the zone of inhibition was6.25±0.94 mm.7.In vivo ulcer healing studies showed that at 7 days,the healing rate of treatment group and control group was 30.4±8.47%and 50.31±6.86%,respectively;at 12 days,the wounds healed in both groups.Conclusions1.The physicochemical properties and structural characteristics of BSP were characterized and analyzed.2.The optimized formula showed sufficient toughness and adhesion,suitable swell-ing rate and pH value.Besides,its crystallinity was enhanced,thermodynamic was stable,and it can maintain flow properties during the reversion from wafer to gel.It reflects the excellent performance of BSP as a biological adhesive material.3.BSP exhibited good biocompatibility and high safety with anti-inflammatory,anti-oxidant and antimicrobial activities,and can promote the healing of oral ulcer,showing that BSP is an ideal adhesive material for oral mucosa and has the potential of accelerating healing for oral ulcer.It is of great significance for the development of drug delivery system for the treatment of oral ulcer.