| Fructus Corni has been used as an important traditional Chinese medicine in China, and it is the dried sarcocarp of Cornus officinalis Sieb. et Zucc. Its action is to benefit liver and kidney and to put restraint on sperm and to solidify collapse. In the recent years, it has been found by research that the extract from Fructus Corni exhibits a number of biological activities, including immunological regulation, reducing blood glucose, antishock, antiarrhythmia, antibiosis, antiinflammatory and so on. Iridoid glycosides including loganin and morroniside are active fraction from Fructus Corni. Loganin exhibits regulating immune function, anti-inflammatory and anti-shock effects and morroniside has been proved to be effective in invigorating stomach and preventing diabetic angiopathies.With the help of dynamic and kinetic mechanisms, pharmacokinetics of traditional Chinese medicine (PTCM) studied absorption, distribution, biotransformation and elimination of active constituents or parts, simple or compound recipes of TCM, and relations of pharmacokinetics and pharmacodynamics of TCM. As one of important contents of modernization of TCM, pharmacokinetics of TCM might be combine the studies of phytochemistry and pharmacodynamics and were beneficial for illuminating efficacy of TCM, identifying therapeutical basis and mechanism of action of TCM. The research of metabolic chemistry profited for discovery of new active constituents and synthesis of new prodrugs.In the present study, A HPLC fingerprint of the total extract from Fructus corni was established and was used for quality control, and the serum fingerprints were investigated after administrated different extracted fractions orally for identifying chemical constituents in rat serum. Then a separation method of loganin and morroniside from Fructus corni was established. Absorption, distribution, metabolism and excretion of loganin and morroniside in rat were investigated for illuminating mechanism of action of theirs. The research provided a significant exploration for pharmacokinetics and therapeutic basis of TCM.Part one Fingerprints and Serum fingerprints of the total extract and its extracted fractions from Fructus corniObjective: A HPLC fingerprint of the total extract from Fructus corni was established and was used to compare its different extracted fractions. The serum fingerprints were investigated in rats after administrated different extracted fractions orally.Methods: The separation was performed on a C18 analytical column with a gradient mobile phase consisting of acetonitrile and 0.15% phosphoric acid at the flow rate of 1.0 mL·min-1. The detection wavelength was set at 217 nm and the temperature of column was kept at 30?C. Total extract, ethyl acetate fraction, n-Butanol fraction and residuary extract and serum samples after oral administration four active parts were analyzed.Results: The similarity among 10 Fructus corni samples were 0.9340.995. Loganin, morroniside, gallic acid and Unknown constituent X were found in total extract from Fructus corni. Composition of ethyl acetate fraction was single and consisted of galic acid and unknown constituent X. Iridoid glycosides, loganin and morroniside, was mainly found in n-Butanol fraction. Ingredients of residuary extract were complicatedly, for example loganin and morroniside and so on, but their content was lower. Loganin, morroniside and unkown constituent X were detected in rat serum, and gallic acid was not.Conclusion: It was shown that the method was in high precision and good repeatability, which provided significant bases on quality control of the total extract from Fructus corni. Some compositions were found in rat serum, so as to illuminate the therapeutical basis of the total extract from Fructus corni preliminary.Part two Study on chemical constituents of Fructus Corni (Preparation of loganin and morroniside reference substance)Objective: To establish a separation method of loganin and morroniside from Fructus corni.Methods: After extracted with hot water and precipitated with alcohol from Fructus Corni, the extract was isolated and purified by macroporous resin, silica gel column chromatography and preparative HPLC. Loganin and morroniside were identified by ultraviolet spectra (UV), 1H nuclear magnetic resonance (1H-NMR), 13C nuclear magnetic resonance (13C-NMR) and mass spectrometry (MS).Results: Three compounds were separated from Fructus Corni and identified as: loganin, morroniside and sweroside. The purity of loganin and morroniside was higher than 98% by normalization method of HPLC.Conclusion: The developed method is simple and at low production cost. The products can be used as reference substance for quality control and drugs for pharmacokinetic study.Part three Studies on absorption, tissue distribution and excretion of loganin in ratsObjective: 1. An HPLC method for determination of loganin in rat plasma was developed for studying the pharmacokinetics of loganin after a single oral administration of loganin and the extract from Fructus corni, and for investigating absolute bioavailability of loganin given orally in rats. 2. An HPLC method was developed and validated for determination of loganin in rat tissues. 3. To investigate excretive characteristics of loganin in rat.Methods: 1. After rats were orally administrated loganin at a dose of 20 mg·kg-1, blood samples were obtained from fossa orbitalis vein according to the specific schedule, 0, 5, 10, 15, 30, 45, 60, 90, 120, 180, 240 and 360 min and collected in heparinized centrifuge tube, respectively. Sample was pretreated by a single-step protein precipitation with methanol. The RP-C18 column (250×4.6 mm,5μm) was used as the stationary phase with the mobile phase consisting of methanol-water (32:68). The flow rate was 1 mL·min-1, the UV detector was set at 236 nm. 2. Rats were randomly assigned to five groups. After oral administration of 20 mg·kg-1 water solution of loganin, heart, liver, lung, spleen, kidney, brain, stomach and small intestine samples were obtained at 15, 45, 90, 180 and 360 min, respectively. Tissue samples were weighed rapidly and put into normal saline solution to remove the blood or content, blotted on filter paper, and then were weighed for wet weight and homogenized in saline solution (600 mg·mL-1). Samples of tissues were prepared based on a simple protein precipitation. Separation of loganin was achieved on a reversed-phase C18 column (250×4.6 mm, 5μm) with a mobile phase consisted of acetonitrile and water (16 : 84, v/v) at a flow rate of 1.0 mL·min-1. The detection wavelength was set at 236 nm and the temperature of column was kept at 30?C. The method was applied to study tissues distribution of loganin in rats after a single administration of loganin at a dose of 20 mg·kg-1. 3. Urine, feces and bile excretion of loganin in rat were investigated by HPLC with protein precipitation and solid phase extraction methods.Results: 1. The calibration curve in plasma was linear over the range of 15.257625 ng·mL-1 and the RSD values of intra-day and inter-day were less than 15%. The recovery of loganin in rat plasma was 93.5%109.3%. After single oral administration of 20 mg·kg-1 loganin and 40 mg·kg-1 extract from Fructus corni to rats, the main pharmacokinetic parameters were as follows: Cmax were 2168.6±455.1 and 1070.6±215.3 ng·mL-1, Tmax were 69.0±20.1 and 51.0±8.2 min, t1/2 were 93.6±36.7 and 99.4±24.6 min, Ke were 0.0085±0.0036 and 0.0073±0.0018 min-1, AUC0-t were 291426.3±75866.8 and 114926.2±32510.7 ng·min·mL-1, AUC0-∞ were 323642.4±83113.7 and 125842.9±36230.6 ng·min·mL -1. The absolute bioavailability of loganin was 13.2% and the relative bioavailability of loganin in the extract from Fructus corni was 19.4%. 2. After a single administration of loganin at a dose of 20mg·kg-1, the highest level was observed in kidney, then in stomach, lung and small intestine. The lowest level was found in brain. The peak levels were attained at 90 min and then declined gradually in most tissues. 3. Loganin was detected in bile but its content was very little and undetected in feces. Drug prototype in urine was completely excreted in 24 h and excretive amount of loganin in 04 h was 81.5%. However, the drug recovery in urine within 24 h was only 5%.Conclusion: 1. Absolute bioavailability of loganin is low. The method is sensitive, simple and suitable for pharmacokinetic parameters study of loganin. 2. Kidney was the major distribution tissue of loganin in rats, and that loganin had difficulties in crossing the blood brain barrier. It was also found there was no accumulation of loganin in rat tissues. 3. Loganin was excreted mainly from urine.Part four Studies on absorption, tissue distribution and excretion of morroniside in ratsObjective: 1. A HPLC method was developed and validated for determination of morroniside in rat plasma and used to study pharmacokinetics of morroniside in rats after a single oral administration of morroniside at different doses. 2. Morroniside was determined in rat tissues after a single oral administration of morroniside at a dose of 20 mg·kg-1. 3. To investigate excretive characteristics of morroniside in rat.Methods: 1. After rats were orally administrated morroniside at a dose of 20 mg·kg-1, blood samples were obtained from fossa orbitalis vein according to the specific schedule, 0, 5, 10, 15, 30, 45, 60, 90, 120, 180, 240 and 300 min and collected in heparinized centrifuge tube, respectively. Samples were prepared based on a simple protein precipitation with methanol. Separation of morroniside was achieved on a reversed-phase C18 column (250×4.6 mm, 5μm) with a mobile phase consisted of acetonitrile-methanol-0.1% formic acid (10 : 10 : 80, v/v) at a flow rate of 1.0 mL·min-1. Chromatograms were monitored at 239 nm and the temperature of column was kept at 25?C. 2. Rats were randomly assigned to five groups. After oral administration of 20 mg·kg-1 water solution of morroniside, heart, liver, lung, spleen, kidney, brain, stomach and small intestine samples were obtained at 15, 30, 60, 120 and 300 min, respectively. Tissue samples were weighed rapidly and put into normal saline solution to remove the blood or content, blotted on filter paper, and then were weighed for wet weight and homogenized in saline solution (600 mg·mL-1). Samples of tissues were prepared based on a simple protein precipitation. The tissue samples were treated with a simple protein precipitation prior to liquid chromatography. Chromatographic condition of morroniside saw 1. 3. Urine, feces and bile excretion of morroniside in rat were investigated by HPLC with protein precipitation and solid phase extraction methods.Results: 1. A good linearity was found in the range of 0.040410.1μg·mL-1. The lower limit of quantification (LLOQ) obtained was 0.0404μg·mL-1 (n=6) using 100μL plasma with an accuracy of–2.62% in terms of relative error (RE) and a precision of 3.2% (RSD). The intra- and inter-day relative standard deviations (RSD) in the measurement of quality control (QC) samples 0.101, 1.01 and 8.08μg·mL-1 ranged from 10.8 to 2.2% and 1.2 to 4.3%, respectively. The accuracy was from 0.08 to 1.22% (RE). Recovery was 99.9%101.8% and RSD was 10.9%3.3%.The analyte was stable in the battery of stability studies. This method was successfully applied to a pharmacokinetic study of morroniside after single oral administration of 10, 20 and 40 mg·kg-1 and intravenous administration of 5 mg·kg-1 morroniside to rats. The main pharmacokinetic parameters were as follows: Cmax were 0.766±0.085,1.292±0.346 and 1.481±0.268μg·mL-1, Tmax were 54±8.2, 60±0 and 60±0 min, t1/2 were 69.4±14.1, 103.9±23.4 and 90.2±32.6 min, AUC0-t were 102.81±17.7, 166.04±30.42 and 193.36±45.56μg·min·mL-1 at dose of 10, 20 and 40 mg·kg-1 morroniside, respectively. Absolute bioavailability of morroniside after single oral administration of 10, 20 and 40 mg·kg-1 was 7.0%,6.1% and 3.6%, respectively. 2. The intra- and inter-day relative standard deviations (RSD) in the measurement of quality control (QC) samples 0.202, 1.01 and 8.08μg·mL-1 were less than 15% and the recoveries of this method were higher than 85%. The highest level was observed in small intestine, then in kidney and stomach. But the level of heart, liver, lung and spleen were lower. Morroniside was undetected in brain. 3. Morroniside was detected in bile but its content was very little and undetected in feces. Drug prototype in urine was completely excreted in 24 h and excretive amount of morroniside in 03 h was 68.5%. However, the drug recovery in urine within 24 h was lower than 2%.Conclusion: 1. Morroniside was absorbed and eliminated rapidly in rat and manifested linear dynamics at 1040 mg·kg-1 range. Absolute bioavailability of morroniside was lower and incomplete. 2. Small intestine, kidney and stomach were major distribution tissues of morroniside in rats, and morroniside had difficulty in crossing the blood-brain barrier. It was also found there was no long-term accumulation of morroniside in rat tissues. 3. Morroniside was excreted mainly from urine.Part five Metabolism of loganin and morroniside in vivo and in vitroObjective: To prepare and identify metabolites by anaerobic culture in vitro and study physiological disposition of loganin, morroniside and their metabolites in rat. At the same time, antidiabetic activity of metabolites was screened for illuminating mechanism of action of loganin and morroniside.Methods: 1. Culture solution of intestinal bacteria were produced from rat feces? and then incubated with loganin and morroniside by anaerobic culture to prepare metabolites of loganin and morroniside. Metabolites of loganin and morroniside were purified by TLC and HPLC and identified by comparison of their ESI-MS, 1D-NMR and 2D-NMR with those of standard and prepared compounds. 2. After taking orally loganin and morroniside, blood, urine, bile, feces, contents of stomach and contents of intestine were pretreated and analyzed by HPLC-PDA. The peaks of loganin, morroniside and its metabolites were validated by comparing their the retention times and the maximum wavelengths of UV spectra. 3. Antidiabetic activity of four metabolites was screened and target point was peroxisome proliferator activated receptors gamma (PPAR gamma).Results: 1. Each two metabolites of loganin and morroniside were isolated and identified, respectively. Mlog-1 was aglycone of loganin, Mlog-2, Mmor-1 and Mmor-2 were validated as new chemical compounds by retrieving Chemical Abstracts (CA). 2. It showed that loganin and morroniside were difficult to be metabolized in stomach and liver. Loganin could be converted to Mlog-1, and then to Mlog-2 by intestinal bacteria. After administrating loganin in rat, loganin could be absorbed in blood and distributed in most tissues, but Mlog-1 and Mlog-2 were undetected in blood. Loganin, Mlog-1 and Mlog-2 were all excreted by urine, but only Mlog-2 by feces and loganin by bile. Morroniside could be transformed to Mmor-1 and Mmor-2 by intestinal bacteria. Morroniside was found in blood but Mmor-1 and Mmor-2 not. Morroniside and Mmor-1 were detected in urine and bile. Mmor-2 could be observed by feces. 3. Mmor-1 and Mmor-2 were found antidiabetic activity.Conclusion: Metabolites of loganin and morroniside prepared in vitro were coincident to those in biological samples. So we can prepare metabolites by anaerobic culture with intestinal bacteria. Metabolic pathway and physiological disposition of loganin and morroniside in rat were presumed on the whole. Morroniside could be transformed to Mmor-1 and Mmor-2 by intestinal bacteria in intestine, and then Mmor-1 and Mmor-2 exhibited antidiabetic effect.
|
PDF Full Text Request