| 1 Purpose and significanceMangiferin [2-C-d-gluco-pyranosyl-1, 3, 6, 7-tetrahydroxyxantone; C19H18O11; Mw, 422.34] is a kind of active flavonoids, a xanthone C-glycoside, and has been reported in various parts of Mangifera indica: fruits, leaves, stem bark; Anemarrhena asphodeloides Bge: leaves, stem bark, roots; Belamcanda chinensis: flower, leaves.Mangiferin (MGF) has poor solubility (<1 mg·mL-1) over the pH range 1.3 to 7.4, due to the large plane molecular structure. The dose of anti-inflammatory for human is about 630mg. It need more than 250 mL volume of aqueous media to dissolve the amount of mangfiferin according Biopharmaceutics Classification System (BCS, abbr.) guidance. BCS is a framework for classifying drug substances based on their aqueous solubility and intestinal permeability. The partition coefficient (P, abbr.) in octanol/water system of MGF is ranging from 0.01 to 2.3, while pH value is from 6.86 to 4. At the same time, the bioavailability of MGF in rats is poor which was evaluating by in situ intestinal perfusion model. Permeability is believed to be the key factors of absorption. Partition coefficient of drug has good correlation with gastrointestinal permeability. Typically, partition coefficient on the range of 100–1000 is required for efficient passive transcellular transport. So it is presume that low bioavailability of MGF may be owing to low permeability. Therefore, MGF is classified as belonging to BCS-4 class, low solubility and low permeability, and mostly encounter serious bioavailability problems. MGF has been reported to have multiple biological effects, including anti-inflammatory, antidiabetic, antioxidant, antitumor, immunomodulatory, anti-allergic, antiviral, antibacterial, etc. The R&D members intended to development MGF as a new drug with anti-inflammatory effect, but we found that MGF had serious absorption problems. To exploit MGF for a new drug, it is essential to ameliorate bioavailability after oral administration. After enhanced the bioavailability of MGF, the given dose of MGF would be reduce ultimately. Currently, some techniques, including salt formation, sulfonate formation, preparation technique to improve solubility, have been carried out to improve the solubility of MGF. But there is no any report on the increase of permeability of MGF.To improve drug's permeability by increasing partition coefficient, acylation is one of the useful and conventional methods for the synthesis of prodrug. The chemical acylation of flavonoids is notregioselective and produce some phenolic-hydroxyl-mask by-products. For shielded the functional hydroxyl group which are responsible for the antioxidant activity of flavonoids, the activity would decrease more or less. However, the enzymatic acylation of flavonoids by lipases with phenolic acids is more regioselective than chemical acylation and may enhance not only their solubility in various media, but also their stability and their antioxidant activity. Lipase is reported to use for the structure modification such as arbutin, isoquercetin, phloridzin, rutin, narigin, etc.Lipase (Lipase, EC 3.1.1.3), three acyl ester hydrolase, is a kind of enzyme catalyzed hydrolysis of natural substrate oil to obtain fatty acids, glycerol and glycerol ester or diester. Lipase is widely used for the reactions of hydrolysis; alcoholysis, esterification, transesterification and ester reverse reaction for substrates (sulfur carboxylates, amides, polyol esters, multi-glycerol triglyceride ester and some hydrophobic ester involved). Lipase Novozym 435 (a form of Candida Antarctica lipase B) is found to be an effective biocatalyst for the acylation of glucose alone with high regioselective to 6-hydroxyl of glucose.Therefore, the enzymatic synthesis of MGF ester, as a kind of prodrug, was investigated on the aim of bioavailability improvement resulted in the enhancing the permeability. In theory, it will provide some ideas for the R&D of BCS 4 drugs.2 Methods and resultsThis work could be divided into several sections, structure modification, Structure identification, Determination of Physical and Chemical Properties and Anti-inflammatory activity assay.(1) MGF was classified as belonging to BCS-4 class via research of biopharmaceutics properties of MGF. The solubility and octanol/water partition coefficient of MGF were very low over the pH range 1.3 to 8.0, solubility changed from 0.16 to 1.44 mg·mL-1, partition coefficient was ranging from 2.313 to 0.05, while pH value was from 6.86 to 4. Moreover, the bioavailability of MGF in rats was low. The permeability of MGF could be increased by synthesis the fat-soluble derivatives.(2) In this part, novel sugar ester prodrugs (formyl MGF, acetyl MGF, propionyl MGF) of MGF, were synthesized by transesterification in non-aqueous medium using commercial immobilized lipase (Novozym 435) as biocatalyst. The HPLC method for reaction process monitoring was established. The molecular weight of these prodrugs was confirmed by HPLC-MS. The enzymatic synthesis was an attractive and economic way for preparation of mono-ester of glycoside, and provided a promising way for grafting acyl group onto glycoside directly.(3) In this part, novel sugar ester prodrug (6′-O-propionyl MGF,PMGF) was synthesized by transesterification in non-aqueous medium using commercial immobilized lipase (Novozym 435) as biocatalyst MGF as acyl acceptor, ethyl propionate as acyl donor without the need of vinyl ester. The HPLC method and purification process of the prodrug was established. The chemical structure of this prodrug was confirmed by HPLC-MS, 1H-NMR, 13C-NM, (g)-HMBC and (g)-HMQC. The effects of the substrate amount, temperature, the nature of the solvent, reaction time and the initial water content were investigated. Novozym 435 retained the highest activity in dioxane. The optimal conditions were the follow: In the 8 mL dioxane containing 30mg MGF and 30mg lipase, adding 1mL ethyl propionate, then the synthesis was performed at 60℃for 20h. Novozym 435 had a high stability. After the lipase continuously used for 15 times, the concentration of product was not changed significantly compared with the first time. Novozym 435 could catalyze the regioselective acylation of 6' hydroxyl group at glucose moiety through the transesterification.(4) In this part, the physicochemical and hydrolysis were evaluated in vitro. Firstly, the equilibrium solubility of PMGF was measured by shake-flask method. The solubility was 0.166±0.02μg·mL-1, and did not decline as a result of fat-soluble increase. Secondly, the octanol/water partition coefficient of PMGF was very low over the pH range 1.3 to 8.0, and was ranging from 21.104 to 0.08. The partition coefficient of PMGF was increased significantly for 4.33 92.36 times compared with MGF. The prodrug was significantly more hydrophilic than MGF. Thirdly, the hydrolysis of MGF and PMGF were studied in hydrochloric acid buffer (pH 2), phosphate buffer (pH 7.4) and plasma solution. MGF showed a high chemical stability in both the aqueous medium of pH 2, pH 7.4 and plasma PBS (pH 7.4). But PMGF was hydrolysis to MGF in vitro. Moreover, three unknown substances were detected in PBS (pH 7.4) medium. It could be due to propionyl transfer to the adjacent hydroxyl of glucose. Summarily, the fat-soluble of PMGF was significantly increased. PMGF was hydrolyzed to prototype MGF in the plasma. It was speculated that the gastrointestinal permeability of PMGF could be increase significantly.(5) In this part, the metabolisms of PMGF and MGF in human intestinal flora in vitro were investigated. Human intestinal flora and PMGF/MGF were incubated under anaerobic conditions. The metabolite were separated and purified by preparative HPLC. PMGF was degraded to PMG within 2 h at the beginning of incubation. This characteristic could be benefit to the absorption of PMGF. Moreover, human intestinal flora could transform PMGF and MGF to aglycone of MGF (norathyriol). Norathyriol was detected at 4h. The absorption mechanism of norathyriol would be planning to investigate.(6) In this part, the anti-inflammatory effect of PMGF was investigated in vitro and in vivo. In vitro, MGF and PMGF (5μg·mL-1) were tested on TNFα, IL-1 and NO production in activated macrophages (RAW264.7 cell line) stimulated with LPS (5μg·ml-1). All the inhibition effects of MGF and PMGF were below 10%. In vivo, the anti-inflammatory effect of PMGF was determined via mice auricular swelling model induced by dimethylbenzene. MGF and PMGF obviously relieved the mice auricular swelling. The effects of middle and low dose of PMGF were better than that of MGF probably due to a higher bioavailability of PMGF attributed to its high permeability.3 ConclusionIn conclusion, on the basis of these results obtain in this work, a kind of high lipophilic MGF ester, 6′-O-propionyl MGF, was obtained. The anti-inflammatory effect of PMGF was better than that of MGF probably due to a higher bioavailability of PMGF attributed to its high permeability. PMGF was demonstrated to be suitable for MGF prodrugs design. In theory, all these works could provide some ideas for the R&D of BCS 4 drugs.4 Innovation(1) For the first time, novel sugar ester prodrugs (formyl MGF, acetyl MGF, propionyl MGF) of MGF, were synthesized by transesterification in non-aqueous medium using commercial immobilized lipase (Novozym 435) as biocatalyst.(2) The fat-soluble MGF prodrug and synergism of anti-inflammatory are achieved.(3) During the course of structure modification of MGF, BCS guidance is applying into the MGF research. The enzymatic synthesis of MGF ester, as a kind of prodrug, was investigated on the aim of bioavailability improvement resulted in the enhancing the permeability.
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