| Sophora tonkinensis Radix et Rhizome(S.Tonkinensis)also known as Guangdougen is the dried root and rhizome of the legume Sophora tonkinensis Gagnep.It is one of the toxic Chinese medicinal materials included in the 2020 Chinese Pharmacopoeia.S.Tonkinensis is cold in nature and bitter in taste.It has the effects of clearing away heat and detoxifying.It has been used for the treatment of sore throat,asthma,jaundice,allergic dermatitis,etc.In addition,modern pharmacological research showed that its extract has a significant anti-inflammatory effect.The preparations of the total alkaloids isolated from S.tonkinensis,such as Ganyanling injection(Shandougen injection),are clinically used for the treatment of chronic hepatitis and liver cancer.However,with the widespread clinical application,numerous cases of the S.tonkinensis toxicity have been reported.The most common case is the neurotoxicity,which is called S.tonkinensis toxic encephalopathy.However,the long-term application can cause brain damage,especially in teenagers.Therefore,the safety issues caused by S.tonkinensis extracts have received extensive attention.In this thesis,using the traditional Chinese medicine S.tonkinensis as the research object,the overall goal is to clarify the contributions of the alkaloids and flavonoids to its anti-inflammatory activity and neurotoxicity.The anti-inflammatory activity and the potential neurotoxicity of different extracts,components(alkaloids,flavonoids)and the isolated main monomer compounds have been studied in vitro and in vivo.In addition,with the representative alkaloids and flavonoids as indicator components,the characteristic fingerprints and index components of S.tonkinensis collected from different regions in China were analyzed to provide quality control of S.main research results are summarized as follows:The water and the 70% ethanol extracts of S.tonkinensis were prepared separately,and the 70%ethanol extract was divided into four fractions(SDG-Fr.1-SDG-Fr.4)with different polarities through the microporous resin(MCI).And their chemical composition was investigated by HPLC with the reference of the standard compounds.It was found that the major chemical constituent of SDG-Fr.2 fraction(40%methanol elution site)is the alkaloids,and the flavonoids is the major component for fraction SDG-Fr.4(100%methanol elution site).Using LPS-stimulated RAW264.7 cells NO release inhibition model,the anti-inflammatory activities of the different extracts,components and main monomer components from S.tonkinensis were evaluated for in vitro anti-inflammation.It was found that the main anti-inflammatory active ingredients are the alkaloids.In addition,for comparison,the anti-inflammatory activity of the flavonoid fraction and the alkaloid fraction were evaluated through the xylene-induced ear swelling model in vivo.The experimental results showed that the dominant contribution for the anti-inflammatory effect is originated from the alkaloids.This result is consistent with the previously reports,which confirmed that the main anti-inflammatory substances of S.tonkinensis are the alkaloids.Through the human neuroblastoma cytotoxicity model,the different extracts,components and main monomer components were also screened for potential neurotoxicity.It was found that both flavonoids and alkaloids are neurotoxic.Through an acute toxicity animal model,a comparative evaluation of the toxicity for the flavonoids and alkaloids was carried out.It was evidenced that the toxicity of alkaloids is much stronger than that of flavonoids,which further confirmed that the main toxic substances of S.tonkinensis are alkaloids.Furthermore,a new quantitative fingerprint method was established by using HPLC,combined with the quantitative determination for the specific standard components of S.tonkinensis.By optimization the separation parameters of HPLC,such as the extraction method,solvent,extraction time,material-to-liquid ratio,the optimum separation conditions for S.tonkinensis extracts were established,as well as for the other Parameters,such as the composition of mobile phase,column type,column temperature,flow rate,detection wavelength,elucidation gradient,etc.In addition,the linearity,limit of quantification,limit of detection,repeatability,stability,and precision of the method were also evaluated.The experimental results showed that the method has reliable repeatability,accuracy,and high precision,and the sample is stable within 24 h.Moreover,the fingerprints of S.tonkinensis collected from different areas of China and the contents of ten components were analyzed by the quantitative fingerprinting method described above.In addition,the similarity of the fingerprints for the different samples of S.tonkinensis was compared according to the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System",Furthermore,based on the components analysis results corresponding ten index compounds,the cluster analysis for the S.tonkinensis was conducted by using SPSS software.The results showed that the similarity of 15 batches of medicinal materials from different areas meets the requirements of the pharmacopoeia,they are S.tonkinensis,the similarity of 3 batches of medicinal materials who are less than 0.9 are not S.tonkinensis,It also shows that this method can be used as the authenticity identification method of S.tonkinensis.The cluster analysis results can divide them into two categories.The experimental data obtained in this study can provide reference for the research on quality control of S.tonkinensis. |
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