MINISTRY OF EDUCATION AND TRAINING SOCIALIST REPUBLIC OF VIETNAM Indepedence – Freedom - Happiness HANOI UNIVERSITY OF PHARMACY o0o DISSERTATION ABSTRACT Introduction Name of candidate: Nguyen Hong Trang Topic of dissertation: “Formulation and application of quercetin phytosome in the dosage form of hard capsules” Specialization: Pharmaceutical technology and Pharmaceutics Code number: 62720402 Name of academic institute: Hanoi University of Pharmacy Summary 2.1 Objectives - Formulation and preparation process of quercetin phytosome - Initial evaluation of in vitro antioxidant and in vivo hepatoprotective effects of quercetin phytosome - Formulation and preparation process of hard capsules containing quercetin phytosome 2.2 Materials, equipments and methods 2.2.1 Materials and equipments - Materials, chemical substances used in the research met the requirement of the Vietnamese Pharmacopoeia or the British Pharmacopoeia (BP) or the U.S Pharmacopeia (USP), other reagents were analytical grade - Research equipments were modern and reliability 2.2.2 Methods Preparation of quercetin phytosome There are two primary methods for preparation of phyto-phospholipid complexes, including solvent evaporation, anti-solvent precipitation Preparation process of quercetin phytosome was summarized in figure 2.1 Quercetin, HSPC and other excipients Quercetin, HSPC and other excipients Dissolved in 20ml ethanol Stirred for complex formation Dissolved in 20ml ethanol Stirred for complex formation Solvent evaporation Precipitation n-hexan Hydration in evaporating flask Solvent evaporation Sonication for formation of phytosomal suspension Dispersion in water 50 ml distilled water Sonication for formation of phytosomal suspension (a) (b) Figure 2.1 The diagram for the preparation process of quercetin phytosome (a) The solvent evaporation method and (b) the anti-solvent precipitation method Preparation of hard capsules containing quercetin phytosome: The manual capsule filling machine was used to fill and close hard HPMC capsules The machine is best suited for small to medium batch size production requirements Evaluation of physicochemical properties of quercetin and phytosome - Characterization of quercetin + Assay: Quercetin content in powder material was determined by HighPerformance Liquid Chromatography or Ultraviolet–visible spectroscopy + Solubility of quercetin in the various mediums was investigated using the classical saturation shake-flask method at the temperature of 25 ± 0,5oC + Octanol-water partition coefficient of quercetin was evaluated by the shake-flask method in a n-octanol-saturated aqueous phase and a water-saturated octanolic phase at the temperature of 25 ± 0,5oC - Characterization of quercetin phytosome + Morphology of the phytosome particles was examined by scanning electron microscope + Mean particle size, particle size distribution (PDI) and zeta potential were determined by the dynamic light scattering (DLS) + Total amount of quercetin in phytosome was quantificated by the HPLC system + Octanol-water partition coefficient and solubility of quercetin phytosome in the various mediums + The amount of quercetin entrapped in the complexion efficiency was determined on the assay of the total amount of quercetin in the phytosome mixture and free quercetin separated by dissolving in ethyl acetate + Dissolution: Using the DT700 Erweka equipment; Medium: pH 1,2 hydrochloric acid containing 20 % v/v of ethanol, 900 mL; Apparatus 2: 100 rpm; Temperature: 37,0 ± 0,5oC + The in vitro release of quercetin from quercetin phytosome was carried out in the Franz diffusion test system (Hanson Research, Germany, 6-Cell Drive System) The 0,2 μm cellulose acetate membrane was used in this study + The interaction between quercetin, soybean hydrogenated phosphatidylcholine, and cholesterol in the phytosome complex was evaluated by analysis of the spectra: IR, 1H-NMR, 13C-NMR, DSC, X-Ray, MS Evaluation of physicochemical properties of capsules containing quercetin phytosome - Characterization of granules containing quercetin phytosome + The granule size: Using the sieves + Moisture content was evaluated by drying the sample to a constant weight, following appendix 9.6, Vietnamese Pharmacopoeia V + Flowability of granules was measured qualitatively using different conventional methods such as angle of repose, bulk and tapped density, Carr’s index and Hausner Ratio This study chose to use the Carr’s compressibility index + Total amount of quercetin in the granules containing quercetin phytosome was assayed by the HPLC system - Characterization of hard capsules containing quercetin phytosome + Form: HPMC capsules in the size of 0, white body and white cap contain light yellow granules Prepared granules have the appropriate pharmaceutical properties e.g uniform particle size, adequate flow characteristics, etc, that allows the easy incorporation of granules into a solid dosage form of good quality + Uniformity of mass was evaluated following Vietnamese Pharmacopoeia V + Assay: Total amount of quercetin in the capsules containing quercetin phytosome was determined by the HPLC method + Dissolution: Using the DT700 Erweka equipment; Medium: pH 1,2 hydrochloric acid containing 0,75 % v/v of Tween 80, 900 mL; Apparatus 1: 75 ± rpm; Time: 45 min; Temperature: 37,0 ± 0,5oC Stability of quercetin phytosome powder and capsules containing quercetin phytosome: Evaluating the change of some quality criteria of phytosome powder and hard capsules containing quercetin phytosome stored under real conditions and accelerated conditions In vitro antioxidant activity of quercetin phytosome: The percentage of antioxidant activity of sample was assessed by DPPH free radical assay In vivo hepatoprotective activity of quercetin phytosome capsules containing quercetin phytosome: The in vivo hepatoprotective effects were evaluated in a classical model of CCl4-induced hepatic injury in mice 2.3 The principal results and conclusion Formulation and preparation process of quercetin phytosome - In this study, quercetin phytosome was prepared by the solvent evaporation technique with the molar ratio of quercetin:HSPC:cholesterol 1:1:0,2 The reaction of quercetin with phopsholipid was carried out at 80oC for hours and the speed of the stirrer was maintained at 50 rpm - Preparation process of quercetin phytosome was developed at the scale of 500 g in a batch - The quality standards for phytosome powder were developed - The stability of quercetin phytosome was performed in two conditions: real conditions, accelerated conditions The research results showed that the obtained phytosome samples were stable during the evaluation period under two experimental conditions (for months) Evaluation of in vitro antioxidant and in vivo hepatoprotective activity of quercetin phytosome - In the in vitro experiment, the antioxidant activity of quercetin phytosome was evaluated and compared with activity of free quercetin Phytosome was unable to increase antioxidant effect on the DPPH assay of quercetin with IC50 values of quercetin and quercetin phytosome were 3,22 µg/ml and 3,30 µg/ml respectively - In the in vivo experiment, the hepatoprotective activity of quercetin phytosome was evaluated and compared with activity of free quercetin In vivo hepatoprotective effect of quercetin in phytosome was improved by reducing ALT, AST in mouse serum, reducing MDA content and increasing GSH content in liver toward normal versus physiological control Formulation and preparation process of hard capsules containing quercetin phytosome - The quercetin phytosome has been successfully applied in the dosage form of hard capsules (each capsule contains 50 mg of quercetin) The dissolution of the obtained capsules was higher than 96 % - The quality standards for the hard capsules containing quercetin phytosome were developed - The stability of capsules containing quercetin phytosome was performed in two conditions: real conditions, accelerated conditions The results showed that the characteristics of the obtained capsules were stable for the duration of the study (six months under the accelerated conditions and 12 months under the real conditions) - The in vivo hepatoprotective effects of the hard capsules containing quercetin phytosome were demonstrated when studied in a classical model of CCl4-induced hepatic injury in mice The obtained results were correlated to results of phytosome powder The scientific advisors Doctoral candidate (Signature and full name) (Signature and full name) Assoc.Prof Vu Thi Thu Giang Prof Pham Thi Minh Hue Nguyen Hong Trang ... Stability of quercetin phytosome powder and capsules containing quercetin phytosome: Evaluating the change of some quality criteria of phytosome powder and hard capsules containing quercetin phytosome. .. process of quercetin phytosome - In this study, quercetin phytosome was prepared by the solvent evaporation technique with the molar ratio of quercetin: HSPC:cholesterol 1:1:0,2 The reaction of quercetin. .. assay of quercetin with IC50 values of quercetin and quercetin phytosome were 3,22 µg/ml and 3,30 µg/ml respectively - In the in vivo experiment, the hepatoprotective activity of quercetin phytosome