| ObjectiveUnder the guidance of traditional Chinese medicine (TCM) theory, a comprehensive and systematic pharmaceutical research of Pothos chinensis (Raf.) Merr. was conducted. Modern separation, analysis and evaluation methods were applied to confirm chemical components, build fingerprints by high performance liquid chromatography (HPLC) and Fourier transform infrared (FTIR) spectroscopy, formulate quality standard draft of Pothos chinensis (Raf.) Merr. and establish quantitative model of moisture contents by near infrared reflectance spectroscopy (NIRS). Preliminary pharmacological studies were carried out on the acute toxicity and the effect of anti-inflammation and analgesia of Pothos chinensis (Raf.) Merr.Methods1. Systematic separation method was adopted to isolate and purify the chemical components of the ethyl acetate part of Pothos chinensis (Raf.) Merr. The structures of the compounds were identified by using thin layer chromatography (TLC), mass spectrometry (MS) and nuclear magnetic resonance (NMR).2. HPLC and IR spectroscopy were used to analyze and identify the fingerprints of Pothos chinensis (Raf.) Merr. from different regions. The similarity of HPLC and IR spectrum of the 24 samples were calculated.3. Microscopic identification was adopted to observe the transaction and the powders while TLC was used to identify β-sitosterol and feruloyltyramine in order to establish the identification methods of Pothos chinensis (Raf.) Merr.. Using HPLC to build the feruloyltyramine content determination method and set limits. Investigating the determination methods of moisture, total ash and extract and setting limits.4. Using NIRS to determinate 24 batches of Pothos chinensis (Raf.) Merr., combined with chemometrics software, the NIRS quantitative models of moisture and extract contents were established.5. Studying on the analgesic effect of water extracts and alcohol extracts by acetic acid writhing test, with the writhing of mice as index, calculating the pain inhibition rate and evaluating the analgesic activity of Pothos chinensis (Raf.) Merr.. By subcutaneous injection of Freund’s complete adjuvant induced adjuvant arthritis (AA) rat model, with the rat paw swelling degree and serum interleukin-1 βlevels as indexes, the anti-inflammatory activity of Pothos chinensis (Raf.) Merr. was evaluated.6. To study the acute toxicity of water extracts of Pothos chinensis (Raf.) Merr., the mice were administrated by gavage. Using the method of Bliss to calculate the median lethal dose (LD50) and the maximum tolerance dose (MTD) of mice, to evaluate the acute toxicity of water extracts of Pothos chinensis (Raf.) Merr.Results1.4 compounds were separated and identified from the ethyl acetate part of Pothos chinensis (Raf.) Merr., respectively syringic acid, vanillic acid, N-trans-feruloyltyramine and β-sitosterol. N-trans-feruloyltyramine and syringic acid were isolated from this plant for the first time.2. The common pattern of HPLC fingerprints was established and 16 common peaks were marked. And the peak 2, peak 3 and peak 11 were identified as vanillic acid, syringic acid and feruloyltyramine. The similarity calculation results of the HPLC fingerprints of 24 batches of Pothos chinensis (Raf.) Merr. showed that similarity of 12 samples were above 0.9,8 samples were between 0.8 and 0.9, and only 4 samples were between 0.7 and 0.8.3. The IR fingerprint of Pothos chinensis (Raf.) Merr. was established, and 15 characteristic absorption peaks were marked. By comparing the overall shape and the characteristic absorption peaks, the purpose of rapid identification can be achieved.4. Established the quality standard of Pothos chinensis (Raf.) Merr.(1) Confirmed the microscopic characteristics of the transaction and the powders of Pothos chinensis (Raf.) Merr.(2) Established TLC methods for the identification of β-sitosterol and feruloyltyramine of Pothos chinensis (Raf.) Merr..(3) Built HPLC method for the determination of the content of feruloyltyramine. Clustering analysis method was applied to analyze the feruloyltyramine content of 24 samples and outlier samples were eliminated. It’s temporarily stipulated that feruloyltyramine content of Pothos chinensis (Raf.) Merr. should not be less than 0.03%.(4) The average value of moisture content of 24 samples was 10.75%, and the average value of total ash was 8.44%. It’s temporarily stipulated that moisture content of Pothos chinensis (Raf.) Merr. should not be more than 13.0%, and the total ash should not be more than 11.0%.(5) Hot dipping method was used to determinate the extract of Pothos chinensis (Raf.) Merr..50% ethanol was chosen to be the extraction solvent. The average value of alcohol soluble extracts of 24 samples was 9.26%. It’s temporarily stipulated that the alcohol soluble extract shshould not be less than 7.0%.5. Established NIRS quantitative models of moisture and extract content. The root mean square error of calibration (RMSEC) of moisture-NIRS model was 0.132; the root mean square error of cross validation (RESECV) was 0.238; the relative error of prediction in validation set was 1.49%. The RMSEC of extract-NIRS model was 0.158; the RMSECV was 0.297; the relative error of prediction in validation set was 0.93%. The models were proven to be credible and accurate, which could be used for rapid determination of the content of moisture and extract.6. Pharmacodynamics studies showed that the water extract and alcohol extract could significantly reduce the writhing times of mice induced by acetic acid pain; the high dose group of both water extract and alcohol extract could significantly reduce the paw swelling of AA model rats; the high, low dose groups of water extract could significantly decrease the level of serum interleukin-1β of AA model rats. The results showed that the water extract of Pothos chinensis (Raf.) Merr. had good analgesic and anti-inflammatory activities, and there was a positive correlation between effect and dose.7. The study of acute toxicity of water extract showed that the LD50 was 207.91g/kg and the MTD was 134.64g/kg. The MTD of mice is one to 269.28 times of the clinical dose of human, which indicated that the acute toxicity of water extract of Pothos chinensis (Raf.) Merr. was low.ConclusionBy studying the chemical components, fingerprints, quality standard, pharmacodynamics and acute toxicity, the overall quality of Pothos chinensis (Raf.) Merr. was evaluated. This research laid a good foundation for the screening of active sites and active ingredients in future, and provided a feasible standard for quality control. This research also provided experimental basis for clinical efficacy and safety of drug, and gave scientific reference for the further development and utilization of Pothos chinensis (Raf.) Merr.. |
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