| Objective:14-deoxyandrographolide(14-DA) is a diterpenoid lactone which is one of the bioactive components isolated from Andrographis paniculata, a traditional herb medical plant. 14-DA is used for treatment of various ailments including possesses multiple pharmacological activities such as antipyresis, anti-inflammation, antibiosis as well as anti-cancer. It’s also used for treatment of liver injury, hypertension and leptospirosis. Our team’s preliminary study found that 14-DA has an inhibitory effect on Lewis lung cancer in vivo. However, the application of 14-DA is limited due to its poor solubility which results in low bioavailability.Liposomes, composed of a lipid bilayer, seem to be an almost ideal drug-carrier system. Both lipophilic and hydrophilic agents can be encapsulated in these nearly spherical lipid vesicles, which has a positive influence on their stability in the bloodstream, their ability to enter various tissues, their interaction with cells, preventing undesirable side effects of drugs and improves the drug therapeutic index. In this paper, we prepared 14-DA liposomes in order to improve its water solubility, thus improving its bioavailability. Methods:A high-performance liquid chromatographic(HPLC) method with UV detection was used for the determination of 14-DA. The shake-flask method was employed to determine the oil-water partition coefficient of 14-DA. Solubility of 14-DA in different media was determined with the saturation method. Liposomes were prepared by thin-film dispersion method. With a single-factor test, three factors, namely ratio of lipid to drug, ratio lipid to cholesterol and thin film preparation temperature, were selected to optimize the composition of 14-DA liposomes. Based on this, the optimal formulation of 14-DA liposomes was determined with central composite design-response surface methodology using encapsulation efficiency, drug loading and particle size as indexes of examination. Ultrafiltration experiment ws carried out to determine 14-DA liposomes encapsulation efficiency. The stability of 14-DA liposomes, 10 mL of which was stored in a penicillin bottle at 4 ℃and 25 ℃in the dark over a period of 30 days, was preliminarily studied by encapsulation efficiency, particle size and pH value. The particle size and Zeta potential were measured by Malvern Zetasizer. The structure of 14-DA liposomes was further observed by transmission electron microscopy. The dynamic dialysis system method was adopted to investigated the release behavior of 14-DA liposomes in 0.3% sodium dodecyl sulfate. Results:1、HPLC method was used for the determination of 14-DA. The chromatographic separation was achieved on a Kromasil ODS column(250×4.6 mm, 5μm) with a Security Guard column(10×4.6 mm, 5μm) filled with the same materials. The temperature was ambient. The mobile phase consisted of acetonitrile–water(40:60, v/v), filtered with 0.45 μm cellulosic Millipore membrane and degassed by ultrasonic before use. It was delivered at an isocratic flow rate of 1.0 mL/min. The detection wavelength was set at 205 nm. The theoretical plate number was not less than 5000 based on 14-DA.2、14-DA was freely soluble in chloroform, soluble in methanol, sparingly soluble in ethanol and acetone, slightly soluble in acetonitrile and ethyl acetate, very slightly soluble in 0.3% SDS and 0.45% SDS, practically insoluble or insoluble in water and 0.1% SDS. Its solubility was stable unless to strong acid and alkali, and it kept relatively constant as the pH ranging form 3.00 to 8.33. The oil-water partition coefficient(LgP) was 2.10.3、Optimized formulation:the compositions(mg) of soybean phospholipids, cholesterol, and 14-DA in the optimized formulation were 150 mg, 48.1 mg and 6.94 mg respectively. They were hydrated into a volume of 25 mL at 39 ℃with the thin-film dispersion method. The encapsulation efficiency of 14-DA liposomes was(78.21±2.50) % and its drug loading capacity was(3.02±0.17) %. The mean particle size was(174.3±3.44)nm and polydispersibility index was 0.206. The mean value of potential was-(37.4±1.56) mV with Malvern Zetasizer. pH value was 6.22. Stored at 25 in the dark over a period of 30 days, its encapsulation efficiency pH value ℃reduced from 73.21% to 58.26%, pH value reduced from 6.22 to 6.04. On the contrary, its mean particle size increased from 174.3 nm to 190.3 nm. It was similar as the temperature was lower from 25 to 4 ℃ ℃, its encapsulation efficiency reduced from 73.21% to 67.98%, pH value reduced from 6.22 to 6.19 and the particle size increased from 174.3 nm to 175.2 nm. Transmission electron microscopy investigations showed that 14-DA liposomes have a spherical or nearly spherical shape. The accumulative release rate of 14-DA liposomes was 72.49% in 24 h, which was less than did that of 14-DA in 12 h(95.78%). Conclusions:As shown above, we established a sensitive and simple method for the determination of 14-DA which had high specificity, recovery rate, reproducibility and precision. The preparation process of 14-DA liposomes was easy to operate and had a high reproducibility. 14-DA liposomes had a high encapsulation efficiency and slow-releasing potential. In adddtion, it seemed more stable at 4.℃... |
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