Benzylisoquinoline alkaloids(BIAs)are a structurally diverse group of plant metabolites renowned for their pharmacological properties.However,sustainable sources for these compounds remain limited.Consequently,researc...Benzylisoquinoline alkaloids(BIAs)are a structurally diverse group of plant metabolites renowned for their pharmacological properties.However,sustainable sources for these compounds remain limited.Consequently,researchers are focusing on elucidating BIA biosynthetic pathways and genes to explore alternative sources using synthetic biology approaches.CYP80B,a family of cytochrome P450(CYP450)enzymes,plays a crucial role in BIA biosynthesis.Previously reported CYP80Bs are known to catalyze the 3′-hydroxylation of(S)-Nmethylcoclaurine,with the N-methyl group essential for catalytic activity.In this study,we successfully cloned a full-length CYP80B gene(St CYP80B)from Stephania tetrandra(S.tetrandra)and identified its function using a yeast heterologous expression system.Both in vivo yeast feeding and in vitro enzyme analysis demonstrated that St CYP80B could catalyze Nmethylcoclaurine and coclaurine into their respective 3'-hydroxylated products.Notably,St CYP80B exhibited an expanded substrate selectivity compared to previously reported wildtype CYP80Bs,as it did not require an N-methyl group for hydroxylase activity.Furthermore,St CYP80B displayed a clear preference for the(S)-configuration.Co-expression of St CYP80B with the CYP450 reductases(CPRs,StCPR1,and StCPR2),also cloned from S.tetrandra,significantly enhanced the catalytic activity towards(S)-coclaurine.Site-directed mutagenesis of St CYP80B revealed that the residue H205 is crucial for coclaurine catalysis.Additionally,St CYP80B exhibited tissue-specific expression in plants.This study provides new genetic resources for the biosynthesis of BIAs and further elucidates their synthetic pathway in natural plant systems.展开更多
Insecticidal activities and active ingredients of Stephania kwangsiensis Lo. were studied for the first time. The results showed that all parts of S. kwangsiensis Lo. had contact activity against brown planthoppers, N...Insecticidal activities and active ingredients of Stephania kwangsiensis Lo. were studied for the first time. The results showed that all parts of S. kwangsiensis Lo. had contact activity against brown planthoppers, Nilaparvata lugens Stal, and the contact activity of methanol extract from root tubers was the highest, with a LD50 value being 1.5794 lag/female. l-roemerine was isolated from root tubers of S. kwangsiensis Lo. and identified, and it was the main active ingredient. l-roemerine had high contact toxicity to brown planthoppers, with a LD50 value being 0.0443 lag/female. Contact toxicity of l-roemerine to brown planthoppers was 7.48 times that of malathion, the convientional chemical insecticide used for controlling brown planthoppers.l-roemerine also had stomach poison activity against brown planthoppers.展开更多
<abstract>Aim: The testicular inhibitory effect of the aqueous fraction of methanol extract of Stephania hernandifolia leaf was studied in male Wistar rats. Methods: The supernatent and the precipitate part of a...<abstract>Aim: The testicular inhibitory effect of the aqueous fraction of methanol extract of Stephania hernandifolia leaf was studied in male Wistar rats. Methods: The supernatent and the precipitate part of aqueous fractions of the methanol extract of the leaf were gavaged separately to rat at a similar dose of 200 mg/mL per 100 g body weight per day for 28 days. After cessation of treatment, various observations were conducted. Results: In both treated groups, there were significant decreases in the relative weights of the sex organs, the testicular key androgenic enzymes activities, the plasma level of testosterone, the number of different germ cells at stage VII of seminiferous epithelial cell cycle and the seminiferous tubular diameter in comparison to the controls. Neither of the parts had somatic, renal and hepatic toxicity. This study suggested that the active molecules present in the aqueous fraction of methanol extract of Stephania hemandifolia leaves might be steroids as indicated by thin layer chromatography using specific staining substance for steroid molecules. Conclusion: In rats, the aqueous fraction of methanol extract of the 5. hemandifolia leaves possesses certain testis-inhibitory substances, which may be steroid-like agents.展开更多
A novel hasubanan alkaloid has been isolated along with the known aknadinine from the fresh roots of Stephania sutchenensis.Its structure has been established as 1—nitroaknadinine from detailed spectral studies and c...A novel hasubanan alkaloid has been isolated along with the known aknadinine from the fresh roots of Stephania sutchenensis.Its structure has been established as 1—nitroaknadinine from detailed spectral studies and confirmed by chemical correlation with aknadinine.The novel alkaloid is the first nitro-hasubanan and also the first example of naturally occurring nitro—alkaloids.展开更多
A novel morphine alkaloid, named gindarudine 1 has been isolated from ethanol extract of Stephania glabra tubers, together with four known alkaloids, palmatine, dehydrocorydalmine, stepharanine, and 8-（4＇-methoxyben...A novel morphine alkaloid, named gindarudine 1 has been isolated from ethanol extract of Stephania glabra tubers, together with four known alkaloids, palmatine, dehydrocorydalmine, stepharanine, and 8-（4＇-methoxybenzyl）-xylopinine. Compound 1 was elucidated as 3,6-O,N-detrimethyl-10-hydroxy-l-methoxy-thebaine by means of spectroscopic data including 2D NMR studies. C 2009 Deepak Kumar Semwal. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.展开更多
Objective To investigate the effect of hydro-ethanolic extraet of Stephania hernandifolia leaves and Aehyranthes aspera roots in composite manner at the ratio of 1：3 on testicular activity in male rats. Methods Rat...Objective To investigate the effect of hydro-ethanolic extraet of Stephania hernandifolia leaves and Aehyranthes aspera roots in composite manner at the ratio of 1：3 on testicular activity in male rats. Methods Rats were divided into 4 groups with 8 animals in each group. The control （group A） received 0. 5 ml of olive oil/100 g body weight orally, other three groups were treated with said extract orally at a dose of 0.4 mg/g （group B） or 0.8 mg/g （group C） or 1.6 mg/g body weight （group D） respectively for 28 d. On 29th day of experiment, the animals were sacrificed. Sperm concentrations in cauda epididymis and biochemical markers like testicular cholesterol, androgenic key enzyme activity, plasma testosterone level seminal fructose level, glutamate oxaloacetate transaminase （GOT） and glutamate pyruvate transaminase （GPT） activities and serum triglyceride levels were measured following standard methods. ResuIts Extract treated animals in all doses resulted in a significant （P〈O. 05）decrease in sperm concentration, testicular androgenic key enzyme activity, plasma testosterone and seminal vesicle fructose levels along with an increase in testieular cholesterol level Animals treated at a dose of O. 8 mg/g body weight showed more promising result without causing any metabolic toxicity compared with other doses. Histological study also supported the biochemical results. The minimum but most effective dose i.e. 0.8 mg/g body weight had an inhibitory effect on implantation focused here by mating experiment.Conclusion The composite efficacy as male contraceptive extract of S. hernandifolia and A. aspera has a potent that may provide clues to the pharmaceutical industries for male contraceptive development展开更多
Stephania Tetrandrae Radix is one of the common traditional Chinese medicines,which has bitter and pungent taste as well as cold properties. It can subside edema,get rid of rheumatism and relieve pain. Therefore,it is...Stephania Tetrandrae Radix is one of the common traditional Chinese medicines,which has bitter and pungent taste as well as cold properties. It can subside edema,get rid of rheumatism and relieve pain. Therefore,it is mainly used for the treatment of rheumatism arthralgia,edema,dysuria,athlete's foot,swollen wet sores and other diseases in traditional Chinese medicine( TCM). Stephania Tetrandrae Radix is mainly composed of dual-benzyl isoquinoline alkaloids,including tetrandrine,fangchinoline and so on. Modern pharmacology research shows that Stephania Tetrandrae Radix and its main components have a wide range of pharmacological activity in the anti-inflammatory,anti-pathogenic microorganisms,anti-tumor,anti-hypertension,anti-arrhythmia,anti-myocardial ischemia,anti-fibrosis,anti-silicosis,inhibiting scar and other aspects,with broad application prospect. Stephania Tetrandrae Radix is often applied with compatibility of other Chinese medicines in clinically,and has achieved obvious effects in the treatment of rheumatoid arthritis,cardiovascular disease,cancer,hypertension,liver ascites and other diseases. There are some representative prescriptions,such as Fangji Fuling decoction,Fangji Huangqi decoction,Jijiao Lihuang pill,Xuanbi decoction,compound Hanfangji granule and so on. In this paper,the pharmacological effects and clinical applications of Stephania Tetrandrae Radix in the past ten years are reviewed,providing the reference for its further development and application.展开更多
[Objectives] To establish a HPLC method for the quantitative determination of cinnamic acid in Stephania longa Lour. [Methods]The chromatographic column: Thermo BDS HYPERSIL C_(18)( 4. 6 mm × 250 mm,5 μm); mobil...[Objectives] To establish a HPLC method for the quantitative determination of cinnamic acid in Stephania longa Lour. [Methods]The chromatographic column: Thermo BDS HYPERSIL C_(18)( 4. 6 mm × 250 mm,5 μm); mobile phase: acetonitrile-0. 1% phosphoric acid( 24∶ 76); flow rate: 1 mL/min; column temperature: 30℃; wavelength: 285 nm. [Results]The cinnamic acid was in a good linear relationship in the range of 0. 021 7-0. 076 μg,the sample recovery rate was 100. 46%,the RSD was 2. 20%,and the content was in the range of 0. 004%-0. 022%. [Conclusions] The method is simple,accurate and reproducible,and can be used for the quality control of S. longa Lour.展开更多
A new artifact bisbenzylisoquinoline,2,2′-N,N-dichloromethyltetrandrine(1),has been ob- tained from the root of Stephania tetrandra and its structure has been advanced on the basis of spectroscopic and chemical evide...A new artifact bisbenzylisoquinoline,2,2′-N,N-dichloromethyltetrandrine(1),has been ob- tained from the root of Stephania tetrandra and its structure has been advanced on the basis of spectroscopic and chemical evidence.展开更多
Objective:To determine the destructive ability of oxocrebanine,an anti-breast cancer active compound obtained from Stephania hainanensis H.S.Lo et Y.Tsoong,on microtubule network,and investigate the effect of oxocreba...Objective:To determine the destructive ability of oxocrebanine,an anti-breast cancer active compound obtained from Stephania hainanensis H.S.Lo et Y.Tsoong,on microtubule network,and investigate the effect of oxocrebanine on microtubule network homeostasis at both molecular and cellular levels.Methods:the EBI site competition method and molecular docking method were used to determine the occupation of the microtubule site of oxocrebanine.Western Blot was used to detect the effect of oxocrebanine on microtubule-associated proteins including STAT3,PAK1,CAMK4,and PKA.Results:The results of EBI site competition assay showed that the binding of EBI toβ-Tubulin covalent fusions produced adducts that appeared in regions of lower molecular weight thanβ-tubulin(ctrl 2).Molecular docking results showed that oxocrebanine could occupy the colchicine site of microtubule proteins.As revealed by Western Blot,the expression of STAT3 protein was decreased after MCF-7 cells have been treated with low,medium,and high concentration of oxocrebanine or the positive drug taxol for 48 h(P<0.01).The expression levels of PAK1 and Camk4 proteins aslo showed significant reductions(P<0.05,or P<0.01).Oxocrebanine also decreased the PKA protein in MCF-7 cells compared to the control group(P<0.01).Conclusions:Oxocrebanine,a ligand that binds at the colchicine site of tubulin,perturbs tubulin polymerization and causes mitosis in MCF-7 cells,thus leading to MCF-7 cell death.Oxocrebanine may promote microtubule dynamics through stathmin by inhibiting the expression levels of STAT3,PAK1,Camk4,and PKA proteins in MCF-7 cells.Oxocrebanine interfers with spindle formation,and ultimately causes mitotic catastrophe in MCF-7 cells.展开更多
BACKGROUND Finding active lead anti-hepatocellular carcinoma compounds from traditional Chinese medicine has important research value.AIM To assess the detailed mechanism of oxocrebanine,a compound separated from the ...BACKGROUND Finding active lead anti-hepatocellular carcinoma compounds from traditional Chinese medicine has important research value.AIM To assess the detailed mechanism of oxocrebanine,a compound separated from the traditional Chinese medicinal plant Stephania hainanensis H.S.Lo et Y.Tsoong,and to evaluate its inhibition of the proliferation of human hepatocellular carcinoma cells via apoptosis and autophagy.METHODS MTT,BrdU labeling,and colony formation assays were used to assess the inhibitory effect of oxocrebanine on the growth and proliferation of human hepatocellular carcinoma Hep3B2.1-7 cells.Flow cytometry was used to detect the effect of oxocrebanine on the apoptosis of Hep3B2.1-7 cells.Western blotting was used to assess the expression of apoptosis-related proteins in Hep3B2.1-7 cells.The aforementioned methods were also used to evaluate the effects of oxocrebanine on cell proliferation,autophagy markers,and autophagy-related protein expression levels after adding autophagy inhibitor 3-mA.Furthermore,to verify the anti-hepatocellular carcinoma effect of oxocrebanine in vivo,a nude mouse model was used to investigate the inhibitory effect of oxocrebanine treatment and its mechanism.Apoptosis was detected using a TUNEL assay and the expression of microtubule-associated protein 1 LC3 in tumor specimens was assessed using immunohistochemistry.RESULTS Oxocrebanine effectively inhibited the growth of Hep3B2.1-7 cells,whilst upregulating the protein expression of cleaved caspase-3,downregulating poly(ADP-ribose)polymerase 1 protein expression,increasing the levels of Bax and Bcl-2 antagonist/killer 1 protein expression,and decreasing the levels of Bcl-2 and myeloid cell leukemia 1 protein expression,which could promote apoptosis in Hep3B2.1-7 cells.Oxocrebanine promoted the transformation of LC3-I to LC3-II in Hep3B2.1-7 cells,suggesting the occurrence of autophagy,whilst the autophagy inhibitor 3-MA could reverse this process.Oxocrebanine was also shown to reduce the phosphorylation levels of the eukaryotic translation initiation factor 4EBP1 and ribosomal protein S6 kinase B1(P70S6K),two downstream effector molecules in the PI3K/Akt/mTOR pathway,inducing autophagy in Hep3B2.1-7 cells.Moreover,the tumor-bearing nude mouse experiment indicated that oxocrebanine effectively inhibited the growth of Hep3B2.1-7 cells in vivo.The results of the TUNEL assay and immunohistochemistry also revealed that oxocrebanine induced apoptosis in vivo and increased the expression level of LC3,an autophagy marker.CONCLUSION Oxocrebanine can inhibit the proliferation of human hepatocellular carcinoma cells by promoting apoptosis and inducing autophagy in vitro and in vivo.展开更多
文摘Benzylisoquinoline alkaloids(BIAs)are a structurally diverse group of plant metabolites renowned for their pharmacological properties.However,sustainable sources for these compounds remain limited.Consequently,researchers are focusing on elucidating BIA biosynthetic pathways and genes to explore alternative sources using synthetic biology approaches.CYP80B,a family of cytochrome P450(CYP450)enzymes,plays a crucial role in BIA biosynthesis.Previously reported CYP80Bs are known to catalyze the 3′-hydroxylation of(S)-Nmethylcoclaurine,with the N-methyl group essential for catalytic activity.In this study,we successfully cloned a full-length CYP80B gene(St CYP80B)from Stephania tetrandra(S.tetrandra)and identified its function using a yeast heterologous expression system.Both in vivo yeast feeding and in vitro enzyme analysis demonstrated that St CYP80B could catalyze Nmethylcoclaurine and coclaurine into their respective 3'-hydroxylated products.Notably,St CYP80B exhibited an expanded substrate selectivity compared to previously reported wildtype CYP80Bs,as it did not require an N-methyl group for hydroxylase activity.Furthermore,St CYP80B displayed a clear preference for the(S)-configuration.Co-expression of St CYP80B with the CYP450 reductases(CPRs,StCPR1,and StCPR2),also cloned from S.tetrandra,significantly enhanced the catalytic activity towards(S)-coclaurine.Site-directed mutagenesis of St CYP80B revealed that the residue H205 is crucial for coclaurine catalysis.Additionally,St CYP80B exhibited tissue-specific expression in plants.This study provides new genetic resources for the biosynthesis of BIAs and further elucidates their synthetic pathway in natural plant systems.
基金the Natural Science Foundation of Guangdong Province(03683).
文摘Insecticidal activities and active ingredients of Stephania kwangsiensis Lo. were studied for the first time. The results showed that all parts of S. kwangsiensis Lo. had contact activity against brown planthoppers, Nilaparvata lugens Stal, and the contact activity of methanol extract from root tubers was the highest, with a LD50 value being 1.5794 lag/female. l-roemerine was isolated from root tubers of S. kwangsiensis Lo. and identified, and it was the main active ingredient. l-roemerine had high contact toxicity to brown planthoppers, with a LD50 value being 0.0443 lag/female. Contact toxicity of l-roemerine to brown planthoppers was 7.48 times that of malathion, the convientional chemical insecticide used for controlling brown planthoppers.l-roemerine also had stomach poison activity against brown planthoppers.
文摘<abstract>Aim: The testicular inhibitory effect of the aqueous fraction of methanol extract of Stephania hernandifolia leaf was studied in male Wistar rats. Methods: The supernatent and the precipitate part of aqueous fractions of the methanol extract of the leaf were gavaged separately to rat at a similar dose of 200 mg/mL per 100 g body weight per day for 28 days. After cessation of treatment, various observations were conducted. Results: In both treated groups, there were significant decreases in the relative weights of the sex organs, the testicular key androgenic enzymes activities, the plasma level of testosterone, the number of different germ cells at stage VII of seminiferous epithelial cell cycle and the seminiferous tubular diameter in comparison to the controls. Neither of the parts had somatic, renal and hepatic toxicity. This study suggested that the active molecules present in the aqueous fraction of methanol extract of Stephania hemandifolia leaves might be steroids as indicated by thin layer chromatography using specific staining substance for steroid molecules. Conclusion: In rats, the aqueous fraction of methanol extract of the 5. hemandifolia leaves possesses certain testis-inhibitory substances, which may be steroid-like agents.
文摘A novel hasubanan alkaloid has been isolated along with the known aknadinine from the fresh roots of Stephania sutchenensis.Its structure has been established as 1—nitroaknadinine from detailed spectral studies and confirmed by chemical correlation with aknadinine.The novel alkaloid is the first nitro-hasubanan and also the first example of naturally occurring nitro—alkaloids.
文摘A novel morphine alkaloid, named gindarudine 1 has been isolated from ethanol extract of Stephania glabra tubers, together with four known alkaloids, palmatine, dehydrocorydalmine, stepharanine, and 8-（4＇-methoxybenzyl）-xylopinine. Compound 1 was elucidated as 3,6-O,N-detrimethyl-10-hydroxy-l-methoxy-thebaine by means of spectroscopic data including 2D NMR studies. C 2009 Deepak Kumar Semwal. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.
文摘Objective To investigate the effect of hydro-ethanolic extraet of Stephania hernandifolia leaves and Aehyranthes aspera roots in composite manner at the ratio of 1：3 on testicular activity in male rats. Methods Rats were divided into 4 groups with 8 animals in each group. The control （group A） received 0. 5 ml of olive oil/100 g body weight orally, other three groups were treated with said extract orally at a dose of 0.4 mg/g （group B） or 0.8 mg/g （group C） or 1.6 mg/g body weight （group D） respectively for 28 d. On 29th day of experiment, the animals were sacrificed. Sperm concentrations in cauda epididymis and biochemical markers like testicular cholesterol, androgenic key enzyme activity, plasma testosterone level seminal fructose level, glutamate oxaloacetate transaminase （GOT） and glutamate pyruvate transaminase （GPT） activities and serum triglyceride levels were measured following standard methods. ResuIts Extract treated animals in all doses resulted in a significant （P〈O. 05）decrease in sperm concentration, testicular androgenic key enzyme activity, plasma testosterone and seminal vesicle fructose levels along with an increase in testieular cholesterol level Animals treated at a dose of O. 8 mg/g body weight showed more promising result without causing any metabolic toxicity compared with other doses. Histological study also supported the biochemical results. The minimum but most effective dose i.e. 0.8 mg/g body weight had an inhibitory effect on implantation focused here by mating experiment.Conclusion The composite efficacy as male contraceptive extract of S. hernandifolia and A. aspera has a potent that may provide clues to the pharmaceutical industries for male contraceptive development
基金Supported by the National Natural Science Fund of China(81360648)
文摘Stephania Tetrandrae Radix is one of the common traditional Chinese medicines,which has bitter and pungent taste as well as cold properties. It can subside edema,get rid of rheumatism and relieve pain. Therefore,it is mainly used for the treatment of rheumatism arthralgia,edema,dysuria,athlete's foot,swollen wet sores and other diseases in traditional Chinese medicine( TCM). Stephania Tetrandrae Radix is mainly composed of dual-benzyl isoquinoline alkaloids,including tetrandrine,fangchinoline and so on. Modern pharmacology research shows that Stephania Tetrandrae Radix and its main components have a wide range of pharmacological activity in the anti-inflammatory,anti-pathogenic microorganisms,anti-tumor,anti-hypertension,anti-arrhythmia,anti-myocardial ischemia,anti-fibrosis,anti-silicosis,inhibiting scar and other aspects,with broad application prospect. Stephania Tetrandrae Radix is often applied with compatibility of other Chinese medicines in clinically,and has achieved obvious effects in the treatment of rheumatoid arthritis,cardiovascular disease,cancer,hypertension,liver ascites and other diseases. There are some representative prescriptions,such as Fangji Fuling decoction,Fangji Huangqi decoction,Jijiao Lihuang pill,Xuanbi decoction,compound Hanfangji granule and so on. In this paper,the pharmacological effects and clinical applications of Stephania Tetrandrae Radix in the past ten years are reviewed,providing the reference for its further development and application.
基金Supported by Project of National Natural Science Foundation(8126067381660701)+1 种基金Scientific Research Innovation Program of Guangxi University of Chinese Medicine(JS201625)Program of Key Laboratory for Purification and Quality Analysis of TCM Extraction in Guangxi Universities(Gui Jiao Ke Yan[2014]No.6)
文摘[Objectives] To establish a HPLC method for the quantitative determination of cinnamic acid in Stephania longa Lour. [Methods]The chromatographic column: Thermo BDS HYPERSIL C_(18)( 4. 6 mm × 250 mm,5 μm); mobile phase: acetonitrile-0. 1% phosphoric acid( 24∶ 76); flow rate: 1 mL/min; column temperature: 30℃; wavelength: 285 nm. [Results]The cinnamic acid was in a good linear relationship in the range of 0. 021 7-0. 076 μg,the sample recovery rate was 100. 46%,the RSD was 2. 20%,and the content was in the range of 0. 004%-0. 022%. [Conclusions] The method is simple,accurate and reproducible,and can be used for the quality control of S. longa Lour.
文摘A new artifact bisbenzylisoquinoline,2,2′-N,N-dichloromethyltetrandrine(1),has been ob- tained from the root of Stephania tetrandra and its structure has been advanced on the basis of spectroscopic and chemical evidence.
基金Natural Science Foundation of Hainan Province(No.820RC776)。
文摘Objective:To determine the destructive ability of oxocrebanine,an anti-breast cancer active compound obtained from Stephania hainanensis H.S.Lo et Y.Tsoong,on microtubule network,and investigate the effect of oxocrebanine on microtubule network homeostasis at both molecular and cellular levels.Methods:the EBI site competition method and molecular docking method were used to determine the occupation of the microtubule site of oxocrebanine.Western Blot was used to detect the effect of oxocrebanine on microtubule-associated proteins including STAT3,PAK1,CAMK4,and PKA.Results:The results of EBI site competition assay showed that the binding of EBI toβ-Tubulin covalent fusions produced adducts that appeared in regions of lower molecular weight thanβ-tubulin(ctrl 2).Molecular docking results showed that oxocrebanine could occupy the colchicine site of microtubule proteins.As revealed by Western Blot,the expression of STAT3 protein was decreased after MCF-7 cells have been treated with low,medium,and high concentration of oxocrebanine or the positive drug taxol for 48 h(P<0.01).The expression levels of PAK1 and Camk4 proteins aslo showed significant reductions(P<0.05,or P<0.01).Oxocrebanine also decreased the PKA protein in MCF-7 cells compared to the control group(P<0.01).Conclusions:Oxocrebanine,a ligand that binds at the colchicine site of tubulin,perturbs tubulin polymerization and causes mitosis in MCF-7 cells,thus leading to MCF-7 cell death.Oxocrebanine may promote microtubule dynamics through stathmin by inhibiting the expression levels of STAT3,PAK1,Camk4,and PKA proteins in MCF-7 cells.Oxocrebanine interfers with spindle formation,and ultimately causes mitotic catastrophe in MCF-7 cells.
基金Supported by National Natural Science Foundation of China,No.82060778the Hainan Provincial Natural Science Foundation of China,No.820RC776+1 种基金the Hainan Province Health Science and Technology Innovation Joint Project,No.WSJK2024MS162the Heilongjiang Province Scientific Research Project of Traditional Chinese Medicine,No.ZHY2024-098.
文摘BACKGROUND Finding active lead anti-hepatocellular carcinoma compounds from traditional Chinese medicine has important research value.AIM To assess the detailed mechanism of oxocrebanine,a compound separated from the traditional Chinese medicinal plant Stephania hainanensis H.S.Lo et Y.Tsoong,and to evaluate its inhibition of the proliferation of human hepatocellular carcinoma cells via apoptosis and autophagy.METHODS MTT,BrdU labeling,and colony formation assays were used to assess the inhibitory effect of oxocrebanine on the growth and proliferation of human hepatocellular carcinoma Hep3B2.1-7 cells.Flow cytometry was used to detect the effect of oxocrebanine on the apoptosis of Hep3B2.1-7 cells.Western blotting was used to assess the expression of apoptosis-related proteins in Hep3B2.1-7 cells.The aforementioned methods were also used to evaluate the effects of oxocrebanine on cell proliferation,autophagy markers,and autophagy-related protein expression levels after adding autophagy inhibitor 3-mA.Furthermore,to verify the anti-hepatocellular carcinoma effect of oxocrebanine in vivo,a nude mouse model was used to investigate the inhibitory effect of oxocrebanine treatment and its mechanism.Apoptosis was detected using a TUNEL assay and the expression of microtubule-associated protein 1 LC3 in tumor specimens was assessed using immunohistochemistry.RESULTS Oxocrebanine effectively inhibited the growth of Hep3B2.1-7 cells,whilst upregulating the protein expression of cleaved caspase-3,downregulating poly(ADP-ribose)polymerase 1 protein expression,increasing the levels of Bax and Bcl-2 antagonist/killer 1 protein expression,and decreasing the levels of Bcl-2 and myeloid cell leukemia 1 protein expression,which could promote apoptosis in Hep3B2.1-7 cells.Oxocrebanine promoted the transformation of LC3-I to LC3-II in Hep3B2.1-7 cells,suggesting the occurrence of autophagy,whilst the autophagy inhibitor 3-MA could reverse this process.Oxocrebanine was also shown to reduce the phosphorylation levels of the eukaryotic translation initiation factor 4EBP1 and ribosomal protein S6 kinase B1(P70S6K),two downstream effector molecules in the PI3K/Akt/mTOR pathway,inducing autophagy in Hep3B2.1-7 cells.Moreover,the tumor-bearing nude mouse experiment indicated that oxocrebanine effectively inhibited the growth of Hep3B2.1-7 cells in vivo.The results of the TUNEL assay and immunohistochemistry also revealed that oxocrebanine induced apoptosis in vivo and increased the expression level of LC3,an autophagy marker.CONCLUSION Oxocrebanine can inhibit the proliferation of human hepatocellular carcinoma cells by promoting apoptosis and inducing autophagy in vitro and in vivo.
