ABSTRACT
@#The main components of the medicinal plant Ilex pubescens Hook. et Arn. are the pentacyclic triterpenoids with various chemical structures. The modification of the triterpenoid skeleton is closely related to a class of key enzymes downstream of the biosynthetic pathway, cytochrome P450 monooxygenase(CYP450). In this study, unigenes with a length of more than 1100 bp were retrieved from the previously obtained Ilex pubescens transcriptome data, and a cytochrome P450 gene(Unigene 0036170)proposed to have the oxidative function of pentacyclic triterpene C-28 was screened through phylogenetic tree analysis. The gene was named as IpAO2. The open reading frame of IpAO2 is 1443 bp, encoding 480 amino acids. RNA was extracted from the tender leaves of Ilex pubescens, and cDNA was obtained by reverse transcription using the RNA as the template. The specific primers AO2-F and AO2-R were designed according to the DNA sequence of IpAO2, and the IpAO2 gene was amplified by PCR using high fidelity enzyme. The IpAO2 gene was ligated into the pEASY cloning vector to obtain pEASY-IpAO2 plasmid. The ligation product was transformed into E. coli Trans1-T1 competent cells and positive clones were screened. The pESC-TRP plasmid was digested with restriction endonucleases to obtain a linearized vector. Primers V-AO2-F and V-AO2-R were designed and the DNA fragment was amplified by PCR using the pEASY-IpAO2 plasmid as template. The DNA fragment was ligated to the vector pESC-TRP to obtain pESC-TRP-IpAO2 recombinant plasmid. The pESC-TRP-IpAO2 plasmid was transformed into a strain of S. cerevisiae capable of efficiently synthesizing amyrin, and the expression of IpAO2 protein was induced with galactose. After extracting the total protein of recombinant S. cerevisiae, Western blot analysis was carried out through the 6×His antigen tag linked to the C-terminus of the target protein. The results showed that the recombinant protein was synthesized in accordance with the expected size. After 7 days of induction and cultivation, the cells of recombinant yeast were collected. The metabolites of cells were extracted and detected by GC-MS after silanization. Through GC-MS analysis of recombinant yeast metabolites, it was found that the protein encoded by the IpAO2 gene can oxidize α-amyrin and β-amyrin to ursolic acid and oleanolic acid, respectively, indicating that IpAO2 is a pentacyclic triterpene C-28 oxidase gene. Meanwhile, the transmembrane domain and protein tertiary structure of the IpAO2 were predicted using the Phyre2 online tool, the results showing that IpAO2 is a transmembrane protein. This study is of great significance to elucidate the biosynthetic mechanism of triterpenoids of Ilex pubescens, and lays a foundation for the further utilization of the metabolic engineering to produce triterpenoids of Ilex pubescens in S. cerevisiae.
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Objective:To establish HPLC-UV fingerprints of Ilex pubescens pieces,and simultaneously determine two components in 46 batches of I. pubescens in pieces of I. pubescens saponin A1 and B1,in order to provide a reference for the quality standard of I. pubescens slices. Method:Methanol was used to extract the I. pubescens saponin samples,and the extracts were measured by HPLC-UV with the absorption wavelength at 210 nm. Kromasil C18 column (4.6 mm×250 mm,5 μm) was used for determining the extracts at a flow rate of 1.0 mL·min-1. The mobile phase condition was acetonitrile-0.1% phosphoric acid aqueous solution with gradient mode. The chromatographic fingerprint similarity evaluation system of traditional Chinese medicine (2012 edition) was used to analyze I. pubescens fingerprints. SPSS 20.0 software was used to cluster the peak area of common peaks. Principal component analysis was performed to reduce the dimension of common peaks. Result:There were great differences between the root and stem parts in I. pubescens fingerprints. The fingerprints of roots and stems of I. pubescens were established respectively,cluster results assorted the roots of I. pubescens into three categories andthe branches of I. pubescens into two categories. The integrity and difference of I. pubescens decoction pieces from different parts and places of origin were compared,and the principal component analysis was performed to screen out the common components that played a decisive role in fingerprint of I. pubescens pieces. And the common peaks were determined. The content of saponin A1 and saponin B1 in Radix I. pubescens were determined. Conclusion:The established I. pubescens fingerprints and content determination methods are simple and suitable. Cluster analysis and principal component analysis are used to screen out the key components of quality control of I. pubescens. The results can provide references for quality control of I. pubescens.
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silica gel column chromatography, Sephadex LH-20 gel column chromatography, medium pressure column chromatography, high pressure flash chromatography, and semi-preparative HPLC, and their structures were elucidated on the basis of physico-chemical constants and spectral analysis. Results: Fourteen compounds were identified as syringin (1), 3, 4-dihydroxyphenyl ethanol (2), oleuropein (3), salidroside (4), oleoside 11-methyl ester (5), (8E)-nuezhenide (6), (8Z)-ligstroside (7), oleoacteoside (8), oleoside dimethyl ester (9), olivil-4'-O-β-D-glucoside (10), (+)-cyclo-olivil-6-O-β-D-glucoside (11), (+)-cyclo-olivil-4'-O-β-D-glucoside (12), ligstroside (13), and wilfordiol B (14). Conclusion: Compounds 2, 4, and 12-14 are obtained from this genus for the first time, and compounds 1, 3, and 5-11 are obtained from this plant for the first time.
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The mice models of blood stasis were established by injecting dexamethasone into the intramuscular of side the thigh for successive 15 days, and giving related drugs via an intragastric administration. Firstly, the method of blocking the bilateral common carotid artery (CCA) was used for 10 minutes, and then perfusion restore for 5 days. Secondly, the method of CCA was used for 30 minutes, and then reperfusion for 24 hours. The whole blood viscosity and plasma viscosity, the activity of NOS and ATPase and the level of NOS, and the content of Glu in the ischemic brain were measured. The morphological changes of brain tissue were observed by eosin (HE) staining technique. The results showed that compared with IPC model group the large doses of the flavonoids could reduce the viscosity of whole blood significantly (P<0.01). The small and medium doses of flavonoids could reduce the whole blood viscosity low-shear obviously (P<0.01). The medium doses of flavonoids could reduce the midst-shear obviously (P<0.05). The large and medium dose of flavonoids could significantly improve the ATP activity (P<0.01). The medium dose of flavonoids could improve the Na⁺-K⁺-ATPase activity significantly (P<0.01). The small dose of flavonoids could improve the Na⁺-K⁺-ATPase activity obviously (P<0.05). The large doses of flavonoids could reduce the content of gluin the ischemic brain significantly (P<0.01). And the others does of flavonoids could reduce the content of gluin the ischemic brain obviously (P<0.05). The large doses of flavonoids could reduce the activity of TNOS and iNOS significantly (P<0.01). The medium doses of flavonoids could reduce the activity of TNOS and iNOS obviously (P<0.05). The small doses of flavonoids could reduce the activity of iNOS obviously (P<0.05). Total flavonoids could obviously or significantly decrease the whole blood viscosity, the activity of NOS and the content of gluin the ischemic brain, increase the activity of ATPase significantly or obviously, could significantly relieve the degree of pathological injury of brain tissue of animal models.
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Objective: To prepare triterpenoid ilexoside O from the roots of Ilex pubescens and to investigate its antithrombotic activity. Methods: Ilexoside O was separated and purified by column chromatography and recrystallization, and was identified by 1H-NMR and 13C-NMR analyses. Afterig administration, the pharmacological effects of ilexoside O were evaluated by clotting time (CT), bleeding time (BT), and the amount of the abdominal aortic blood clots induced by FeCl3 in rats, respectively. Results: In comparison with placebo group, ilexoside O showed the obvious effects on BT (P<0.01). The low-dose group (25 mg/kg) of ilexoside O showed no obvious effect on CT (P≤0.05), but the mid-(50 mg/kg) and high-(100 mg/kg) dose groups obviously increased CT of mice (P<0.05); Ilexoside O obviously decreased the amount of the abdominal aortic blood clots in the induction of FeCl3 in rats (P<0.01). Conclusion: Ilexoside O shows obvious anticoagulant effect and could decrease the amount of the abdominal aortic blood clots, and its related mechanism is one of the problems which need to be further studied.
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OBJECTIVE: To develop the high-performance thin-layer chromatographic (HPTLC) fingerprints of triterpenoid from Ilex pubescens and compare the fingerprints similarity with its related species, such as Ilex rotunda and Ilex asprella. METHODS: The separation was performed on pre-coated HPTLC GF254 silica-gel plate stationary phase with trichloromethane-ethyl acetate-methanol-water-formic acid(3:22:5:2:0.2) as solvent system and 10% sulfuric acid alcoholic solution as color development reagent. The common pattern of HPTLC fingerprints were obtained through Chromafinger2005 solution software, and authentication and quality assessment were analyzed by similarity and Principle Commonent Analysis. RESULTS: The common pattern of the pileus of Ilex pubescens consists of 13 characteristic peaks. There are notable differences among the fingerprints of different parts of Ilex pubescen, and those between Ilex pubescens and its related species. CONCLUSION: The different parts of Ilex pubescens can be distinguished by comparison of the HPTLC chromatographs. The other relatives can be easily differentiated from Ilex pubescens too. Copyright 2012 by the Chinese Pharmaceutical Association.
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Objective To establish a method for the determination of bacterial endotoxin in Ilex pubescens compound injection(LPCJ) .Methods According to China Pharmacopoeia (second part,2000),interference test was carried out to detect the endotoxins in LPCJ with limulus lysate agents (?was 0.25 EU/mL).Results Inhibition action was found in bacterial endotoxin test for LPCJ and in the 10-,20-,30-and 40-fold diluted liquid of LPCJ.It was proved that the maximum noninterference concentration was the 50-fold dilution.The inspection results are the same as that of rabbit pyrogen test.Conclusion The 50-fold diluted LPCJ is suitable for the determination of bacterial endotoxin and can be used as the quality control standard for its preparation.