Genetic diversity of protopine-6-hydroxylase in three medicinal Papaver plants / 中国中药杂志

Sanguinarine is the main active component of the Papaver plants, and protopine-6-hydroxylase(P6 H), involved in the sanguinarine biosynthetic pathway, can oxidize protopine to 6-hydroxyprotopine. The investigation on the diversity of P6 H genes in the medicinal Papaver plants contributes to the acquirement of P6 H with high activity to increase the biosynthesis of sanguinarine. Five P6 H genes in P. somniferum, P. orientale, and P. rhoeas were discovered based on the re-sequencing data of the Papaver species, followed by bioinformatics analysis. With the elongation factor 1α(EF-1α), which exhibits stable expression in the root and stem, as the internal reference gene, the transcription levels of P6H genes in roots and stems of the Papaver plants were detected by real-time fluorescent quantitative PCR. As indicated by the re-sequencing results, there were two genotypes of P6H in P. somniferum and P. orientale, respectively, and only one in P. rhoeas. The bioinformatics analysis showed that the P6 H proteins of the three Papaver plants contained the conserved domain cl12078, which is the characteristic of p450 supergene family, and transmembrane regions. The existence of signal peptide remained verification. Real-time fluorescent quantitative PCR results revealed that the transcription level of P6 H in roots of P. somniferum was about 1.44 times of that in stems(α=0.05). The present study confirmed genetic diversity of P6 H in the three medicinal Papaver plants, which lays a basis for the research on the biosynthesis pathway and mechanism of sanguinarine in Papaver species.

Protopine inhibits the growth of hepatocellular carcinoma through a mitochondrially mediated apoptosis pathway / 药学学报

Hepatocellular carcinoma (HCC) is a serious threat for human health, the incidence of HCC in China accounts for more than 50% worldwide. There is an urgent need to develop novel anticancer agents for the treatment of HCC patients. Here we characterized the inhibitory effect and the molecular mechanism of protopine on HCC cancer cells. The results of a CCK-8 assay indicated that protopine displays anticancer activities on HCC cells. Flow cytometry and JC-1 staining confirmed that treatment with protopine decreased the mitochondrial membrane potential and induced apoptosis in HCC cells. Western blot analysis showed that protopine was able to increase protein expression in the mitochondrial apoptotic pathway; the level of cytochrome C, apoptotic protease activating factor-1 (Apaf-1), Bax, cleaved-poly ADP-ribose polymerase (cleaved-PARP), cleaved-caspase-3, and cleaved-caspase-9 were increased while the expression of Bcl-2 was suppressed significantly. An in vivo study revealed that protopine significantly suppressed the growth of tumors in nude mice bearing HepG2 cells. Administration of protopine intraperitoneally at a concentration of 50 mg·kg-1 inhibited tumor growth by 72.46%. Animal experiments were carried out according to the Regulation of the Animal Ethics Committee of Southwest Medical University. This study provides preliminary evidence that there is potential to develop protopine as a lead compound for the treatment of HCC.

Location, distribution, and quantification of myenteric plexus neurons of the jejunum of quails fed with different levels of commercial Macleaya cordata extract / Localização, distribuição e quantificação dos neurônios do plexo mioentérico do jejuno de codornas de postura alimentadas com diferentes inclusões do extrato comercial de Macleaya cordata

ABSTRACT: Coturniculture has been promising, progressing from a subsistence to a technical activity due to its quick production, low breeding investment, and rapid economic return. After the restriction of antimicrobials as growth promoters, some studies aimed to evaluate alternative products that would make the farming of healthy birds viable without impacting their performance, with commercial Macleaya cordata extract being one of these substitutes. The functions of the gastrointestinal tract are coordinated mainly by the enteric nervous system, and the myenteric plexus is responsible for the reflex control of contractile activities of the external muscles. Thus, this study located and demonstrated the distribution of the myenteric plexus, quantifing the total population of myenteric neurons (Giemsa+) and the subpopulation of myenteric nitrergic neurons (NADPH-d+), and evaluated the effects of commercial Macleaya cordata extract on these populations of quail jejunum neurons. A total of 240 one-day-old female laying quails were distributed into four treatments, with four repetitions of 15 birds each. The test groups (T1, T2, and T3) were treated with commercial Macleaya cordata extract throughout the experimental period using the following doses: T1 - test group, basal diet added with 150 ppm of the extract in the feed; T2- test group, basal diet added with100 ppm of the extract in the feed; T3 - test group, basal diet added with 50 ppm of the extract in the feed; and T4 - control group, basal diet with no added extract. The study included histological analysis, Giemsa+, and NADPH-d+ myenteric neuron staining. The results showed that the myenteric plexus is located between longitudinal layer fibers and in the transition region between the longitudinal and circular layers of the muscular tunic, with the myenteric population organized into ganglia and isolated in the region of neuronal fiber bundles. The commercial Macleaya cordata extract showed no quantitative changes in the myenteric Giemsa+ population and myenteric NADPH-d+ subpopulation, however, the groups that consumed the extract showed greater NADPH-d+ neuron activity compared to the control group, implying that the food remained longer in the intestinal lumen, therefore, enabling greater nutrient use and resulting in increased productive performance.

RESUMO: A coturnicultura tem apresentado características promissoras, deixando de ser uma atividade de subsistência e ocupando patamares tecnificados devido a sua precocidade produtiva, baixo investimento de criação e rápido retorno econômico. A partir da restrição da utilização de antimicrobianos como promotores de crescimento, estudos foram direcionados com o objetivo de se avaliar produtos alternativos que viabilizassem a criação de aves saudáveis, sem comprometer seu desempenho, sendo o extrato comercial da Macleaya cordata um destes substitutos. As funções do trato gastrintestinal são coordenadas principalmente pelo sistema nervoso entérico, sendo o plexo mioentérico responsável pelo controle reflexo das atividades contráteis da musculatura externa. Desta forma, o presente trabalho teve como objetivo localizar e demonstrar a distribuição do plexo mioentérico, quantificar a população total de neurônios mioentéricos (Giemsa+), e a subpopulação de neurônios mioentéricos nitrérgicos (NADPH-d+), além de avaliar os efeitos do extrato comercial da Macleaya cordata sobre estas populações de neurônios do jejuno de codornas. Foram alojadas 240 codornas de postura, fêmeas, com um dia de idade, distribuídas aleatoriamente em quatro tratamentos, com quatro repetições de 15 aves cada. Os grupos testes (T1, T2 e T3) foram tratados com extrato comercial de Macleaya cordata durante todo o período experimental conforme as doses indicadas, sendo: T1 - grupo teste, com dieta basal adicionada de 150 ppm do extrato na ração; T2 - grupo teste, com dieta basal adicionada de 100 ppm do extrato na ração; T3 - grupo teste, com dieta basal adicionada de 50 ppm do extrato na ração, e T4 - grupo controle, com dieta basal isenta do extrato. Foram realizadas análise histológica e a marcação dos neurônios mioentéricos Giemsa+ e NADPH-d+. Os resultados demonstraram que o plexo mioentérico está localizado entre as fibras do estrato longitudinal, e na região de transição entre os estratos longitudinal e circular da túnica muscular, estando a população mioentérica organizada em gânglios, e também isoladamente na região dos feixes das fibras neuronais. O extrato comercial da Macleaya cordata não alterou quantitativamente os neurônios da população mioentérica Giemsa+ e da subpopulação mioentérica NADPH-d+, mas os grupos que consumiram o extrato apresentaram maior atividade dos neurônios NADPH-d+ em relação ao grupo controle, permitindo inferir que o alimento permaneceu maior tempo no lúmen intestinal e, portanto, possibilitou um maior aproveitamento dos nutrientes, podendo refletir em melhor desempenho produtivo.

Relationship Between Characters and Main Components of Chelidonii Herba Pieces / 中国实验方剂学杂志

Objective::To determine the relationship between the characters and the main components of Chelidonii Herba pieces. Method::The main components of the samples were determined by HPLC, and the characters of Chelidonii Herba Pieces were evaluated by sensory evaluation. The correlation between the characters and components was calculated by correlation formula. Result::The content of 6 components, such as chelidonine, was determined by HPLC. Based on the result of sensory evaluation, there was a certain correlation between the characters and the components. Character descriptions that " some have visible white powder, some have white pubescence, and sometimes they have visible yellow florets" had a very low similarity with total alkali, indicating that these characters and total alkali content was not related. The similarity between the description of " hollow stem" had a high similarity with total alkali content, indicating that the amount of stem was related to the total alkali content. The character description of " more broken leaves" was negatively correlated with total alkaloids in the similarity, which indicated that the content of total alkaloids was less when there were more leaves(or more broken leaves), otherwise, the content of total alkaloids was relatively higher. Conclusion::The established HPLC method is simple and feasible. This study objectively quantifies the descriptions of Chelidonii Herba pieces characters, correlates them with the main components of Chelidonii Herba pieces, and then preliminarily judges the quality of Chelidonii Herba pieces according to the appearance of the characters, which provides a theoretical basis for the identification of Chelidonii Herba pieces in the market by experience, and ideas for the study of the characters of other traditional Chinese medicines.

HPLC fingerprint and multicomponents determination of Corydalis Decumbentis Rhizoma / 中草药

Objective: To establish the HPLC fingerprint and multicomponents determination of Corydalis Decumbentis Rhizoma, and provide a scientific basis for the improvement of its quality standards. Methods The separation was performed on a chromatographic Diamonsil C18 column (250 mm × 4.6 mm, 5 μm), with acetonitrile-0.2% acetic acid (adjusting pH to 6.0 with triethylamine) as the mobile phase for gradient elution. Volume flow rate was 1.0 mL/min. Column temperature was 35 ℃. Injection was 10 μL and the detection wavelength was 280 nm. The fingerprints of 15 batches of Corydalis Decumbentis Rhizoma were established and evaluated by the similarity evaluation system of TCM (version 2012A), which were divided into two categories by clustering analysis and principal component analysis. Meanwhile, the content of protopine, palmatine, bicuculline and tetrahydropalmatine was determinated. Results:The fingerprint of Corydalis Decumbentis Rhizoma was established. There were 10 common peaks in the fingerprint. Protopine, palmatine, bicuculline and dl-tetrahydropalmatine were separated with good linearity relationships (r > 0.999 9). The average recovery rates of the investigated compounds were 97.12%, 100.09%, 98.53%, and 99.71%, respectively. Conclusion:HPLC fingerprint combined with multicomponents determination can provide the reference for the quality evaluation of Corydalis Decumbentis Rhizoma.

Inhibitory Effects of Protopine on the Proliferation of Human Hepatic Stellate Cells HSC-LX 2 and Its Mechanism Study / 中国药房

OBJECTIVE： To investigate inhibitory effects of protopine on the proliferation of human hepatic stellate cells HSC-LX2 and to explore its mechanism preliminarily. METHODS： MTT assay was used to detect the effects of 25， 50， 100， 200， 400 and 500 μmol/L protopine on the proliferation of HSC-LX2 cells. The inhibitory effect of cell proliferation was calculated. HSC-LX2 cells were divided into control group （1640 medium containing 5％ fetal bovine serum）， protopine low-concentration， medium-concentration and high-concentration groups （100， 200， 400 μmol/L）. After treated for 24 h. The apoptotic rate of the cells was detected by flow cytometry. RT-PCR was used to determine the mRNA expression of α-SMA， Collagen Ⅰ， Collagen Ⅲ， MMP-2 and TIMP-1 in cells. The protein expressions of α-SMA， Collagen Ⅰ， Collagen Ⅲ and MMP-2 were detected by Western blot. RESULTS： The inhibitory rates of 25， 50， 100， 200， 400 and 500 μmol/L protopine on proliferation HSC-LX2 cells were 0， 6.9％， 18.7％， 34.2％， 48.9％， 53.9％， respectively. Compared with control group， mRNA expression of Collagen Ⅰ， TIMP-1 and protein expression of α-SMA were decreased significantly in protopine low-concentration， medium-concentration and high-concentration groups， while protein expression of MMP- 2 was increased significantly， with statistical significance （P＜0.05 or P＜0.01）. Apoptotic rate of HSC-LX2 cells and mRNA expression of MMP-2 were increased significantly in protopine medium-concentration and high-concentration groups， mRNA expression of α-SMA and Collagen Ⅲ， protein expression of Collagen Ⅰ and Collagen Ⅲ were decreased significantly， with statistical significance （P＜0.05 or P＜0.01）. CONCLUSIONS： Protopine can induce the apoptosis of HSC-LX2 cells and inhibit their cell proliferation， and reduce the expression of a-SMA， Collagen Ⅰ， Collagen Ⅲ and TIMP-1， and increase the expression of MMP-2.

Fingerprint profiling of Macleaya cordata fruits from different regions / 中草药

Objective To establish the fingerprint profiling of fruits of Macleaya cordata and study the method for its quality evaluation. Methods The fingerprint profiling of M. cordata fruits from different regions was established using high performance liquid chromatography (HPLC) based on the local standard which was the determination method of the content of protopine, allocryptopine, sanguinarine, and chelerythrine from Changsha of Hunan Province in 2009. The principal component analysis (PCA) and cluster analysis (CA) were performed to explore the correlation among the common fingerprint peaks, origins, and quality of M. cordata fruits. Results Eleven common fingerprint peaks were identified in the fingerprint profiling of chemical constituents of M. cordata fruits from different regions. M. cordata fruits produced from eight areas were classified into two classes by PCA and CA method, and there were five common peaks, including peak 5, 7, 8, 9, and 11 with significant contribution on the regional difference of the fingerprint. Also, common peak 6 was the right peak as the reference peak because of its less variation, appropriate retention time and intensity. Conclusion The fingerprint profiling of chemical constituents of M. cordata fruits established in this study has good precision, repeatability, and stability, which can be used to evaluate the quality of fruits of M. cordata from different producing areas.

Optimization of microwave processing technology for vinegar Corydalis Rhizoma by response surface methodology / 中草药

Objective To optimize the microwave processing technology of vinegar Corydalis Rhizoma. Methods On the basis of single factor test, the index was evaluated by total rating of content of tetrahydropalmatine, protopine and the total alkaloid, which were determined by HPLC and UV spectrophotometry method, four factors (fire, stuffy time, processing time and dosage of vinegar) were studied by response surface, the microwave processing technology of vinegar Corydalis Rhizoma was optimized by response surface methodology. Results The optimal parameters of microwave processing technology were as follows: microwave power of 70%, stuffy time of 1.5 h, microwave time of 2.6 min, vinegar dosage of 27.5%, the content of tetrahydropalmatine, protopine, and total alkaloid were 0.112 4%, 0.041 8%, and 0.85%. Conclusion Microwave processing can be used as a processing method to enrich the traditional processing technology.

Quality control for alkaloids of Corydalis Rhizoma based on quantitative analysis on multi-components with single marker / 中草药

Objective: To establish an HPLC method for simultaneous assay of seven main active constituents (protopine, coptisine, palmatine, dehydrocorydaline, D-tetrahydro jatrorrhizine, tetrahydropalmatine, and corydaline) in Corydalis Rhizoma, and on this basis, establishing a methodology of quantitative analysis on multi-components by single marker (QAMS) to validate the feasibility of method and technical adaptability of quality control applications for Corydalis Rhizoma. Methods: Taking seven components in Corydalis Rhizoma as indicators, two correction methods were used to establish the relative correction factor (fk/s) between each component and tetrahydropalmatine. Then the correction factor was used to calculate the amount of each component in Corydalis Rhizoma and finally to achieve this method. In the meantime, the external standard method was used to measure the above seven components to compare the difference between the calculated and measured value of the two fk/s, and to validate the correctness and adaptability of QAMS. Results: The methodology of QAMS which was used to evaluate the seven kinds of alkaloids in Corydalis Rhizoma was established; There was no significant difference between the data calculated by QAMS in different columns and instruments and the values measured by the external standard method. Conclusion: The QAMS method for measuring the components of protopine, coptisine, palmatine, dehydrocorydaline, D-tetrahydro jatrorrhizine, tetrahydropalmatine, and corydaline in Corydalis Rhizoma is reliable and accurate, it could be used to control the quality of crude drugs and herbal pieces of Corydalis Rhizoma.

Simultaneous determination of seven alkaloids in Cuyanhusuo Granule by RP-HPLC / 中草药

Objective: To establish HPLC method for the simultaneous determination of protopine, palmatine hydrochloride, berberine hydrochloride, dehydrocorydaline, tetrahydropalmatine, tetrahydroberberine, and corydaline in Cuyanhusuo Granule. Methods: The analysis was performed on Ultimate AQ-C18 column (250 mm×4.6 mm, 5 μm) by gradient elution of acetonitrile-0.1% phosphoric acid (adjust to pH 6.0 with triethylamine) (10:90). The UV detection wavelength was set at 280 nm and the flow rate was 1.0 mL/min. Results: The linear ranges of protopine, palmatine hydrochloride, berberine hydrochloride, dehydrocorydaline, tetrahydropalmatine, tetrahydroberberine, and corydaline were 6.8-119.0 (r=0.9999), 24.38-426.65 (r=0.9999), 8.88-155.40 (r=0.9999), 77.66-1359.05 (r=0.9999), 41.4-724.5 (r=0.9999), 6.70-117.25 (r=0.9999), and 25.50-446.25 ng (r=0.9999). The average recoveries (n=6) were 98.2% (RSD=2.0%), 99.6% (RSD=2.8%), 100.2% (RSD=1.3%), 99.0% (RSD=2.2%), 100.8% (RSD=2.6%), 98.7% (RSD=2.5%), and 97.7% (RSD=2.2%), respectively. Conclusion: This method is simple and rapid, and can be used for the quality control of Cuyanhusuo Granule with satisfactory separation and repeatability.

Determination of Protopine in Rat Brain Tissues by RRLC-ESI/Q-TOF-MS Method / 中草药·英文版

Objective: To analyse the quantification of protopine from Corydalis Decumbentis Rhizoma (CDR) extract in brain tissues of rats. Methods: A rapid, sensitive, and accurate analytical method based on rapid resolution liquid chromatography (RRLC) coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (Q-TOF-MS) was developed for the quantification of protopine in brain of rats. A simple liquid-liquid extraction process was employed for the sample preparation. Chromatographic separation was achieved using 1.8 μm porous particle columns. Results: The calibration curve of protopine was linear in the range of 12-897 ng/mL. The relative standard deviations of intra- and inter-day precision values were less than 10%. The extraction recoveries were 96.4%, 99.6%, and 98.5%, for protopine at the concentration of 598.0, 119.6, and 12.0 ng/mL, respectively, and internal standard (1.27 μg/mL) was (98.60 ± 0.02)%. Conclusion: The validated method is successfully applied for the determination of protopine in brain tissue of rats after ig administration of CDR extract. © 2014 Tianjin Press of Chinese Herbal Medicines.

Effect of Different Processes on the Contents of Active Ingredients in Corydalis / 中国中医药信息杂志

Objective To explore the effect of different processes on the contents of active ingredients in Rhizoma Corydalis. Methods Processes of vinegar-boiling, vinegar-frying and alcohol-frying were used to fresh corydalis, boiled corydalis and purchased corydalis. Then the method of extraction by ultrasound with water and by reflux with methanol were used to extract the sample. The contents of Protopine, Tetrahydropalmatine and Dehydrocorydaline in corydalis was determined by RP-HPLC. Agilent TC-C18 (25 mm?4.6 mm, 5 ?m) column was used at 35 ℃, with acetonitrile-0.1% phosphonic acid (pH 5.3 with triethylamine) (28∶72) as mobile phase. The flow rate was 1 mL/min and detection wavelength was set at 280 nm. Results In the sample extracted by water, the contents of active ingredients in processed products were higher than in corydalis. And in the sample extracted by methanol, it also has the tendency that the contents of active ingredients in processed products were higher than in corydalis. The content of Dehydrocorydaline in fresh corydalis which was processed by vinegar-boiling, vinegar-frying and alcohol- frying was high. The content of Tetrahydropalmatine in purchased corydalis which was processed by water-boiling at production place was high. Conclusion The method of determining active ingredients in Rhizoma Corydalis was established. It was convenient and the result was accurate. The process technology that fresh corydalis was boiled by water and then processed or fresh corydalis was processed in production place can be used.

Regulation of glutamate level in rat brain through activation of glutamate dehydrogenase by Corydalis ternata

When treated with protopine and alkalized extracts of the tuber of Corydalis ternata for one year, significant decrease in glutamate level and increase in glutamate dehydrogenase (GDH) activity was observed in rat brains. The expression of GDH between the two groups remained unchanged as determined by Western and Northern blot analysis, suggesting a post-translational regulation of GDH activity in alkalized extracts treated rat brains. The stimulatory effects of alkalized extracts and protopine on the GDH activity was further examined in vitro with two types of human GDH isozymes, hGDH1 (house-keeping GDH) and hGDH2 (nerve-specific GDH). Alkalized extracts and protopine activated the human GDH isozymes up to 4.8-fold. hGDH2 (nervespecific GDH) was more sensitively affected by 1 mM ADP than hGDH1 (house-keeping GDH) on the activation by alkalized extracts. Studies with cassette mutagenesis at ADP-binding site showed that hGDH2 was more sensitively regulated by ADP than hGDH1 on the activation by Corydalis ternata. Our results suggest that prolonged exposure to Corydalis ternata may be one of the ways to regulate glutamate concentration in brain through the activation of GDH.

Effect of protopine on contraction and intracellular free calcium concentration of smooth muscle of thoracic aorta in rats / 第三军医大学学报

Objective To study the effect of protopine (Pro) on the intracellular free calcium ions of the smooth muscular cells of the thoracic aorta in rats. Methods The procedure of calcium absence-calcium addition was designed to observe the changes of the level of calcium ions in the strips of the smooth muscle of the thoracic aorta indirectly. The concentration of calcium ions in the smooth muscle of the thoracic aorta was determined with Fura-2/AM loaded SMCs. The elevation of calcium ions was induced with norepinephrine (NE). The concentration of potassium ions was observed in the presence of Pro. Results Pro significantly inhibited the NE-induced transient contract of the smooth muscle of the thoracic aorta in calcium-free medium and long-lasting contraction after the addition of calcium ions in a concentration-dependent manner. In Fura-2/AM loaded SMCs, Pro (50 ?mol/L or 100 ?mol/L) exerted no effect on the resting calcium ions but obvious effect on the NE-induced and high potassium level-induced elevation of calcium ions. In the presence of extracellular calcium ions, Pro (50 ?mol/L) decreased the NE-induced elevation of calcium ions but showed no effect on potassium-induced elevation of calcium. Pro (100 ?mol/L) significantly decreased NE-induced and high potassium level-induced elevation of calcium ions. Conclusion Pro reduces NE-induced or high potassium level-induced elevation of calcium ions in the smooth muscle of the thoracic aorta through its effect on calcium release and/or calcium influx.

Determination of Protopine in Rhizoma Corydalis Decumbentis Capsule by HPLC / 中药新药与临床药理

Objective To develop a HPLC method for the determination of protopine in Rhizoma Corydalis Decumbentis Capsules. Methods A HPLC was performed on a Hypersil ODS column( 4.6? 250 mm, 5 ? m) . The mobile phase was methanol - 0.1 % water solution of triethylamine (65∶ 35). Determination wavelength was 290 nm and flow rate was 1.0 mL/min. The theoretical plates should over 4000 according to the content of protopine. Results A good linearity was shown in the range of 0.3168～ 1.5840 ? g, r=0.9996. The average recovery was 100.6 % , RSD=2.68 % . Conclusion The method is simple, effective and can be used for the quality control of Rhizoma Corydalis Decumbentis Capsules.

Determination of protopine and tetrahydropalmatine in Corydalis Decumbens (Thunb.) Pers. by HPLC / 中成药

Objective:To establish the content determination method of the main alkaloids protopine and tetrahydropalmatine in Corydalis decumbens (Thunb.) Pers. Methods: HPLC was used to determinate the main alkaloids protopine and tetrahydropalmatine in Corydalis decumbene (Thunb.) Pers. Supelcosil LC 18 DB Column was used, mobile phase consisted of Methol NaAc/HAc(75∶25 pH5.0) and detection wavelength at UV 285 nm. Results: Linearity of this method was good with the average recoveries, 98.04% and 99.63%. Conclusion: This method is accurate, reliable with good separability and reproducibility, it can be applied as standard for the control of Corydalis decumbens (Thunb.) Pers.

Distrubution and excretion of protopine in rats by RP-HPLC method / 中国药理学通报

AIM To study the distrubution and excretion of protopine in rats. METHODS Reversed phase high performance liquid chromatographic method (RP HPLC) was developed for determining the level of protopine in rats. The analytical column were packed with 5 ?m C 18 . The mobile phase was a mixture of methanol, water and 10% acetic acid (80∶20∶2), in which the pH was modulated to 5 6 with 15% ammonia. Protopine biological samples were isolated well, in which two extraction with ether under basical condition and an extraction with 0 02 mol?L -1 sulfuric acid were performed, respectively. The content of protopine in the biological sample was measured by an UV detector at 285 nm. The distrubution and excetion of protopine have been investigated in rats after intravenous administration 10 mg?kg -1 . RESULTS Protopine distrubuted in many tissues after iv a dose of 10 mg?kg -1 . The higher level of protopine was found in lung, kidney, spleen and brain, and the highest was observed in lung at 5, 15 minutes after administration. However the top level tissue was testicle at 3 h, which may be due to small blood circulation. The excretion of the parent compound in urine was 36 87% of dose, but the excretion of the parent compound in feces and bile was less than 1% of dose. Plasma protein binding was less than 5%. CONCLUSION The distrubution of protopine is extensive and the parent compoud was mainly excreted by urine and plasma protein binding was low.