Research Of Pharmacological Effects Of Houttuynine Analogues

Houttuynine analogues—octanoyl acetal sodium sulfite, dccanoy] acetal sodium sulfite, dodecanoyl acetal sodium sulfite, tetradecanoyl acetal sodium sulfite, palmitoyl acetal sodium sulfite, octadecyl acetal sodium sulfite were synthesized. Based on different hydrophobicities of Houttuynine analogues, the analytic method of them both in vivo and in vitro, metabolism rule and residue in vivo and effects on the function of macrophages of mice both in vivo and in vitro , activity of SOD in serum and adjuvanticity on BSA were studied. Taking the molecular structure of octanoyl acetal sodium sulfite as a starting point, a series of derivatives, viz., methyl octanoyl acetal sodium sulfite, ethyl octanoyl acetal sodium and butyl octanoyl acetal sodium sulfite, were synthesized by adding alkyl to the a-site of two carbonyls and their immunological enhancement were studied.The ultraviolet absorption of Synthetical Houttuynine analogues was weak in water. But Synthetical Houttuynine analogues can be hydrolyzed and their ultraviolet absorption can be changed under acid or alkali conditions. Under alkali conditions, Diheptadecanoyl acetaldehyde sodium hydrosulfite was hydrolyzed to Diheptadecanoyl acetaldehyde, and then Diheptadecanoyl acetaldehyde was transformed to its enol form by the isomerization; the enol form contained conjugated double bond, so its ultraviolet absorption was strengthened obviously and the most absorbing wavelength is around 285 nm. According to it. a HPLC analytical method of Houttuynine analogues with low concentration was established in this test.The result of HPLC analysis using mobile phase mixed with tetrabutyl ammonium hydroxide showed that the peaks of all Houttuynine analogues can be well separated. With the augmentation of C-number of alkyl of these six compounds, their hydrophilic activities decreased, retention time increased and they can be separated better. The range of their retention time varied from 7.23min to 12.83min.The simulating physiological condition test in vivo indicated that after entering into blood circulatory system, Houttuynine analogues were hydrolyzed to yeile decanoyl aldehyde and sodium sulfite. Decanoyl aldehyde can be adsorbed onto cell membranes and SO₃^2- can penetrate through cell membranes and get into cells.After intramuscular according to the dose of 25mg/kg body weight, the distribution of Houttuynine analogues in mice body conformed one-compartmental open model. There was favorable consistency among the metabolism rules of this kind of compounds in mice. After intramuscular injection, the peak time of the concentration of HOU-C_n in blood was short (1.311±0.001hr) , the velocity of their absorption by mice tissues was rapid, the difference of half time (5.907±0.002hr) and the rate of elimination among all Houttuynine analogues was little.With the increasing of the carbon chain, their hydrophobicity strengthened, apparent distribution volume (Vd) value increased (0.294～0.377) , peak- concentration in blood (Cmax) and area under the curve(AUC) value decreased. The Cmax (56.853mg/L～72.829 mg/L) , AUC value (557.672～724.072) and bioavailability (F) of the six Houttuynine analogues were high. After administration for 48h, excluding in muscle , the residual amount of HOU-Cn in the tissues such as brain, heart, liver and kidney can be detected. With the increasing of carbon chain of HOU-Cn, the residual amount of HOU-Cn in brain tended to increase.Taking tetradecanoyl acetal sodium sulfite (HOU-C14) as sample of the six analogues, according to literatures, the range of test concentrations was set from 1.5mg/kg to 36mg/kg. Lysozgme activity in serum increased with the augmentation of concentrations of HOU-C14 and it was the highest at the concentration of 24 mg/kg. Based on above result, each Houttuynine analogy in each different research items was tested at the concentration of 12mg/kg, 24mg/kg and 36mg/kg, respectively. The follow results also proved that using this range of concentration to study was available.Thymus gland index of normal mice behaved an improvement trend in the experiment, especial HOU-C14, the thymus gland index of it at three concentrations were all much higher than it in blank control group and levamisole positive control group, and thymus gland index of other analogues at three concentrations all also increased. Spleen index differed greatly. The spleen indexes of Houttuynine analogues except HOU-C14 at three concentrations were higher than blank control group. The results were inconsistent with other literatures. It may be caused by different concentrations of tested drugs and different administrations. While the multiplication of spleen lymphocytes caused by HOU-C14 can maintain for a longer time than other tested analogues.Six analogues made obvious effects on phagocytosing percentages and phagocytosing indexes of macrophages of mice. The phagocytosing percentage and phagocytosing index of a fixed Houttuynine analogy reached the highest value at the concentration of 24mg/kg. But the phagocytosing percentage of HOU-C16 and the phagocytosing index of HOU-C10 were the highest at the concentration of 36mg/kg. The phagocytosing percentages and phagocytosing indexes of different Houttuynine analogues at a fixed concentration (24mg/kg) increased with the increasing of the length of the aliphatic chain and increaseing of aliphatic hydrophobicity; while the phagocytosing percentage and phagocytosing index decreased with increasing of the length of the aliphatic chain and aliphatic hydrophobicity when the length of the aliphatic chain exceeded a certain range (phagocytosing percentage of H0U-C14, phagocytosing index of HOU-C10). but their phagocytosing percentages and phagocytosing indexes were still higher than them in blank control group.The effect of octanoyl acetal sodium sulfite on shape of enteroceli macrophages in vitro was dependent on concentration and time. When the concentration of octanoyl acetal sodium sulfite in inoculum was lower than 50mg/L, macrophages membrane possessed stability and shape of macrophages can not be changed within 24 hours. While, when the concentration exceeded 75mg/L, macrophages were dissolved.The effects of the tested six Houttuynine analogues on phagocytic activity relating with concentrations. Within the range of tested concentrations (12mg/kg～36mg/kg) , phagocytic actibity strengthened with the augmentation of concentrations, but the capability of enhancing phagocytic actibity was weakened at high concentration. The effects of different analogues on phagocytic actibity of macrophages at a fixed concentration in vitro consisted with the one in vivo. The capability of enhancing phagocytosing function was strengthened with the increasing of the length of the aliphatic chain and increasing of aliphatic hydrophobicity; but the capability was weakened when the length of the aliphatic chain exceeded a certain range(C-14). The phagocytosing percentages and phagocytosing indexes of macrophages among all tested groups differed indistinctively in vitro and the correspond values in vitro was one time higher than the one in vivo.The level of SOD and lysozyme in serum of mice and activity of lysozyme of macrophages in vitro were increased by the test six Houttuynine analogues. The level of SOD and lysozyme of Houttuynine analogues at a fixed concentration (24mg/kg) increased with increasing of the length of the aliphatic chain and increasing of aliphatic hydrophobicity; but the enhancement of the level of SOD and lysozyme was weakend when the length of the aliphatic chain exceeded a certain range(C-14). The level of lysozyme of macrophages in inoculum was much higher than it in serum.Suspended BSA-Cn antigens were produced by mixing three Houttuynine analogues (dodecanoyl acetal sodium sulfite, tetradecanoyl acetal sodium sulfite, palmitoyl acetal sodium sulfite) with BSA independently. BSA-Cn antigens showed obvious immunogenicity in New Zealand rabbits after two immunizations. Absorption value at 450nm (A450) of anti-BSA antibodies in serum was the highest when rabbits had been immunized for 14 days and the A450 of antibodies had descended after 24 days. The A450 of three anti-BSA antibodies groups increased straightly and they all had increased by 6 to 20 times after two immunizations. A450 of BSA-C14 group was two times higher than BSA group after 14 and 24 days and the changing rule was nearly the same as BSA group after 14 days. The typical adjuvanticities of these three Houttuynine analogues were found in this test, especial tetradecanoyl acetal sodium sulfite. Houttuynine and its analogues are the typicalβ-two carbonyl compounds. Houttuynine and its analogues possesse two aliveα-carbon atoms, according to the theory of organic chemistry ,hydrogen atom in a-carbon atom is easily substituted; theα-carbon atom between two carbonyl groups is more reactive than the other one and hydrogen atom in former is more easily substituted. Based on this theory, in this research, taking the molecular structure of octanoyl acetal sodium sulfite as a starting point, a series of derivatives, viz., methyl octanoyl acetal sodium sulfite, ethyl octanoyl acetal sodium and butyl octanoyl acetal sodium sulfite, were synthesized by alkylation to hydrogen atom inα-carbon atom to obtain methyl, ethyl and butyl.Synthesized productions having carbonyl group, aldehyde group, the lone electron pair and double bond and purity of synthesized productions were confirmed by determination of melting point, 2,4-dinitrophenylhydrazine reaction, reaction of sodium bisulfite affixture with fuchsin aldehyde and method of ultraviolet scanning. Synthesized productions possessed the same functional groups of-OH, CH₃-,-CH₂-and-C=O et al, which was conformed by IR with the strong absorbing belts at the ranges of from3800 to 3500 cm^-1, from 1700 to 1500cm^-1 and from 1560 to 1480cm^-1. The chemical structure ofα-C octanoyl acetal sodium sulfite derivatives , viz., methyl octanoyl acetal sodium sulfite, ethyl octanoyl acetal sodium and butyl octanoyl acetal sodium sulfite were conformed by ¹H NMR, ¹³C NMR and mass spectra.The research results indicated that octanoyl acetal sodium sulfite and its threeα-C dericants all incresed the level of SOD in serum and lysozyme secrected by macrophages in vitro. With the increasing of the number ofα-C alkyl, the augmentation of level of SOD and lysozyme was weakened by tested compounds. The effect of butyl octanoyl acetal sodium sulfite on the level of SOD in serum and lysozyme secrected by macrophages approximately equaled to the effect of control group. Comparing the effect of three dericants on the level of SOD in serum and lysozyme secrected by macrophages with which of octanoyl acetal sodium sulfite, the augmentation of dericants descended straightly with the increasing of the number ofα-C alkyl, which was quite different from the above result that the immunoregulative activities of Houttuynine analogues were enhanced with increased in the length of the aliphatic chain. It indicated that the compounds produced by structure qualification to straight-chain and branched chain may make different effects on immunization of animals.