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Qualitative and quantitative analysis of 2-(2-phenylethyl) chromones in sodium chloride(NaCl)-treated suspension cells of Aquilaria sinensis was conducted by UPLC-Q-Exactive-MS and UPLC-QQQ-MS/MS. Both analyses were performed on a Waters T3 column(2.1 mm×50 mm, 1.8 μm) with 0.1% formic acid aqueous solution(A)-acetonitrile(B) as mobile phases at gradient elution. MS data were collected by electrospray ionization in positive ion mode. Forty-seven phenylethylchromones was identified from NaCl-treated suspension cell samples of A. sinensis using UPLC-Q-Exactive-MS, including 22 flindersia-type 2-(2-phenylethyl) chromones and their glycosides, 10 5,6,7,8-tetrahydro-2-(2-phenylethyl) chromones and 15 mono-epoxy or diepoxy-5,6,7,8-tetrahydro-2-(2-phenylethyl) chromones. Additionally, 25 phenylethylchromones were quantitated by UPLC-QQQ-MS/MS. Overall, the rapid and efficient qualitative and quantitative analysis of phenylethylchromones in NaCl-treated suspension cells of A. sinensis by two LC-MS techniques, provides an important reference for the yield of phenylethylchromones in Aquilariae Lignum Resinatum using in vitro culture and other biotechnologies.
Subject(s)
Chromones , Sodium Chloride , Chromatography, Liquid , Flavonoids , Tandem Mass Spectrometry , ThymelaeaceaeABSTRACT
Abstract@#Saposhnikovia divaricata, a perennial herb belonging to the family Umbelliferae, is widely distributed in many provinces of Mongolia. The dried root of Saposhnikovia divaricata has been used for the treatment of arthritis and as a painkiller in Mongolian folk medicine. Moreover, its dried root (Radix Saposhnikoviae) is used as a Chinese herbal medicine for the therapy of immune system, nervous system, and respiratory diseases. According to phytochemical and pharmacological studies, the main ingredients of Saposhnikovia divaricata are chromones, coumarins, acid esters, and polyacetylenes. These compounds indicate anti-inflammatory, antioxidant, analgesic, antiproliferative, and immunoregulatory activities. Cimifugin is an active ketone ingredient from Saposhnikovia divaricate, Rhizoma cimicifugae. Cimifugin has been reported to have bacteriostatic and antiviral effects. Studies have reported that cimifugin inhibits allergic inflammation by reducing the levels of cytokines. The aim of this review is to provide extensive information on the traditional use, ethnopharmacology, phytochemistry, pharmacology mechanism of action, and health products from Saposhnikovia divaricata .
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The calibration of chromone reference extract(CRE) was conducted and a quality control method of Saposhnikoviae Radix(SR) was established based on CRE. Meanwhile, the quality control system of SR was improved and the feasibility of using reference extract as a substitute for single reference substance in quality control of Chinese medicine was discussed. In this study, the content of the prepared CRE was calibrated with prim-O-glucosylcimifugin, cimifugin, 4'-O-β-D-glucosyl-5-O-methylvisamminol, and secO-glucosylhamaudol as indicators. Subsequently, an HPLC analytical method was developed to determine the content of four chromones in 20 batches of SR samples based on the CRE with known content as the standard substance. T-test was used for the comparison of the determination results of the two methods(single chemical component and CRE as reference substances, respectively), and the P values of prim-O-glucosylcimifugin, cimifugin, 4'-O-β-D-glucosyl-5-O-methylvisamminol, and sec-O-glucosylhamaudol were 0. 16,0. 39, 0. 14, and 0. 42. The results demonstrated that there was no significant difference between the two methods. This study initially verified the feasibility that the CRE could be used as a substitute for single reference substance in quality control of SR. In conclusion,this study is expected to provide a scientific basis and a new research model for the application of reference extract in the quality control of Chinese medicine.
Subject(s)
Apiaceae , Calibration , Chromatography, High Pressure Liquid , Chromones , Drugs, Chinese Herbal , Quality ControlABSTRACT
2-(2-Phenylethyl) chromones are only distributed in a few plant species and have many bioactivities such as neuroprotection, antibacterial and anti-inflammatory effects. They are the characteristic and main active components of agarwood. They are expected to have broad medicinal application value. In this paper, the research progress of the biological activities such as neuroprotective activity, anti-tumor cytotoxic activity, antibacterial activity and anti-inflammatory activity, as well as the structure-activity relationship and biosynthetic pathway of 2-(2-phenethyl) chromone compounds were reviewed and prospected. It provides a reference for screening the pharmaceutical 2-(2-phenethyl) chromone compounds and analyzing their biosynthetic pathways. Some problems in the current biosynthesis researches are also pointed out here.
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Objective: To analyze the main chemical constituents of traditional Chinese medicine compound Chenxiang Huaqi Pills by using UPLC-Q-TOF/MS technology. Methods: The separation was performed on Phenomenex Kinetex C18 column (100 mm×4.6 mm, 2.7 μm), and the gradient elution of acetonitrile-0.1% formic acid was used as mobile phase at a flow rate of 0.8 mL/min. The data was collected by the positive and negative ion modes using Q-TOF/MS and ESI source. The main chemical constituents of Chenxiang Huaqi Pills were identified according to the exact molecular mass, the cleavage fragments of MS/MS, the literature data, and the reference control. Results: A total of 73 chemical components were separated and identified in Chenxiang Huaqi Pills, including 36 flavonoids, 16 2-(2-phenylethyl) chromones, 7 triterpenoid saponins, 2 sesquiterpene lactones, and 12 other components. Conclusion: This study showed that UPLC-Q-TOF/MS technology provided a simple, rapid, and accurate method for the identification of chemical constituents in Chenxiang Huaqi Pills, which provided a new technology method for the pharmacological basis and quality control of Chenxiang Huaqi Pills.
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Objective: HPLC-MS was used to analyze the characteristic constituent of Aquilariae Lignum Resinatum, providing basis for the establishment of quality evaluation system of agarwood. Methods: The analysis was carried out on a Dionex-Acclaim 120 C18 column (250 mm × 4.6 mm, 5 μm). The mobile phase consisted of acetonitrile and water-acetic acid (1000.5) with the flow rate of 0.4 mL/min at 254 nm, and the separation was performed at 26 ℃. MS experiments were performed using an electrospray ionization tandem mass spectrometry mainly in positive-ion mode. The cluster analysis was performed by SPSS software. Results: A total of 14 common peaks of flidersia chromones were characterized by HPLC-MS analysis, and seven of them were identified by comparing their retention times and MS spectra with reference compounds. The 10 batch of samples were divided into five categories by cluster analysis. The result indicated that great difference existed between agarwood produced from living tree and dead tree, and the relationship between constituent and production place of “Huang-Shu” agarwood was obvious. Conclusion: The analytical method for common flidersia chromones established in this paper could be used to evaluate the quality of agarwood.
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Objective To study the chemical constituents of agarwood from Aquilaria crassna. Methods The compounds were isolated and purified by various column chromatographic techniques. Their structures were identified on the basis of spectral data and physicochemical properties. And the compounds were tested for α-glucosidase inhibitory activity by pNPG method. Results One new sesquiterpenoid along with two known chromones, which were determined as methyl crassicid (1), 8-chloro-6-hydroxy-2-(2- phenethyl) chromen-4-one (2), 8-chloro-6-hydroxy-2-[2-(4-methoxyphenyl) ethyl] chromen-4-one (3), were obtained from the ethyl acetate extract obtained from 95% ethanol extract of agarwood from A. crassna. Conclusion Compound 1 is a new sesquiterpenoid named sesquiterpenoid, which exhibited α-glucosidase inhibitory activity with IC50 value of 121.8 μg/mL.
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Objective: To establish a method for the simultaneous determination of four chromones in Saposhnikoviae Radix by multi-components with single marker ( QAMS) .Methods:An HPLC analysis was used to determine the contents of 4′-O-β-D-glucosyl-5-O-methylvisamminol , cimifugin and sec-O-glucosylhamaudol in Saposhnikoviae Radix.Prim-O-glucosylcimifugin was chosen as the single maker.The contents of 4 chromones in 10 batches of samples were determined by both external standard method and QAMS .The reliability and feasibility of the method were evaluated by the comparison of the quantitative results between the external standard meth -od and QAMS.Results:The relative correction factor (RCF) was perfect.The results calculated by the single marker were consistent with the results from the external standard method .Conclusion:The method with single marker is accurate and feasible to evaluate the quality of Saposhnikoviae Radix.
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Objective: To study the non-alkaloid constituents from stems and leaves of Ochrosia elliptica. Methods: The chemical constituents from O. elliptica were separated and purified by silica gel, ODS, Sephadex LH-20 gel column chromatography, and preparative HPLC. Their structures were identified by physicochemical properties, spectroscopic analysis, as well as comparisons with the data in literature. Results: Fifteen compounds were isolated from the ethyl acetate fraction of 90% ethanol extract from O. elliptica, which were identified as hedyotisol A (1), (-)-(7R,7'R,7″S,8S,8'S,8″S)-4',4″-dihydroxy-3,3',3″,5-tetramethoxy-7,9',7',9-diepoxy-4,8″-oxy-8,8'-sesquineolignan-7″,9″-diol (2), 8-hydroxypinoresinol (3), lyoniresinol (4), balanophonin (5), formononetin (6), vestitol (7), mucronulatol (8), (3R)-violanone (9), 6-hydroxy-2-(2-phenylethyl) chromone (10), 6-hydroxy-2-[2-4'-(methoxyphenyl) ethyl] chromone (11), 4-hydroxyacetophenone (12), dehydrovomifoliol (13), loliolide (14), and 4,5-dihydroblumenol A (15). Conclusion: All compounds are isolated from the genus Ochrosia Juss. for the first time.
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Medicinal plants in Cimicifuga L. contain triterpenoid glycosides, phenolic acids, chromones and other ingredients, showed biological activities of antitumor, anti-inflammatory, antiviral, anti-nucleoside transport, anti-osteoporosis, anti-oxidant, antidepression, etc, and have been used in the treatment of perimenopausal syndrome. This paper reviews the advances in studies on chemical constituents cimicifuga, focuses on the research progress of biological activities and clinical applications to provide a reference for further development of medicinal plants in Cimicifuga L.
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Objective To study the characteristic 2-(2-phenylethyl)chromone components of endophytic fungal strain of Aquilaria sinensis by solid culture. Methods The compounds were isolated by various chromatographic methods such as silica gel, reverse-phase silica gel, Sephadex-LH20 column chromatography as well as crystallization. Results Seven 2-(2-phenylethyl)chromone analogues were isolated from the solid culture of Botryosphaeria rhodina A13. Their structures were established by spectral data as well as physicochemical properties, and identified as 6-hydroxy-7-methoxy-2-(2-phenylethyl)chromone (1), 6,7-dimethoxy-2-(2-phenylethyl) chromone (2), (5S,6R,7S,8R)-2-(2-phenylethyl)-5,6,7,8-tetrahydrchromone (3), 6-hydroxy-2-(2-phenylethyl)chromone (4), 4′-hydroxy-2-(2-phenylethyl)chromone (5), 6-methoxy-2-phenethyl-4H-chromen-4-one (6), and 6-methoxy-2-(4′-methoxy-phenethyl)-4H-chromen-4-one (7). Conclusion All of the compounds are isolated for the first time from the genus Botryosphaeria. This research opens up a new vista to produce the characteristic components of agarwood by endophytic fungi.
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Fourteen compounds were isolated from the stem of Angelica polymorpha. On the basis of spectral data, these compounds were identified as isoimperatorin (1), phellopterin (2), bergapten (3), xanthyletin (4), cnidilin (5), geijerine (6), (−)-3'-acetyl hamaudol (7), 7-demethylsuberosine (8), dehydrogeijerin (9), (−)-hamaudol (10), (+)-visamminol (11), divaricatol (12), scopoletin (13), and decursidate (14), respectively. Among them, compounds 4 - 6, 8, 9, 13, and 14 were isolated for the first time from A. polymorpha. Dehydrogeijerin (6) and geijerin (9) were isolated for the first time from genus Angelica. All isolates tested for inhibitory activity against acetylcholinesterae. Compounds 1 to 13 showed acetylcholinesterase inhibitory activity with IC₅₀ values ranging from 1.4 to 37.5 µM.
Subject(s)
Acetylcholinesterase , Angelica , Cholinesterase Inhibitors , Chromones , Coumarins , ScopoletinABSTRACT
Objective: To develop an HPLC-DAD method for the simultaneous determination and comparative analysis on the contents of prim-O-glucosylcimifugin, 4′-O-β-D-glucosyl-5-O-methylvisamminol, cimifugin, sec-O-glucosylhamaudol, psoralen, imperatorin, bergapten, and xanthotoxin in the roots of Saposhnikovia divaricata from Longxi areas. Methods: The extracts were obtained by methanol reflux method and the contents of the eight compounds were determined by HPLC-DAD. The mobile phase, consisting of acetonitrile-water, was programmed for a gradient elution. The flow rate was 1.0 mL/min, the detection wavelength was 254 nm, and the column temperature was 25°C. Results: Excellent linearity with correlation coefficents (r) of 0.9992-0.9999 was obtained. The average recoveries of the eight compounds were 96.2%-104.1% and all RSD values were less than 3%. The contents of the four coumarins in the roots of S. divaricata were much less than those four chromones. The contents of the four coumarins in the roots of Carum carvi and Peucedahum ledebourielloides were more than those in S. divaricta, while no chromones were detected except sec-O-glucosylhamaudol in P. ledebourielloides. Conclusion: The method appears to be simple, accurate, and well reproducible, which could be used for the simultaneous determination of the above-mentioned eight compounds in S. divaricata. According to the above analysis, C. carvi and P. ledebourielloides could not be used as the succedaneum of S. divaricata on the basis of the eight compound contents.
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Objective To investigate the effects of signal pathway inhibitors PD98059 and LY294002 on cell proliferation, apoptosis, expressions of phosphorylated extracellular signal-regulared kinase (p-ERK) and phosphorylated protein kinase B ( p-Akt) in endometrial carcinoma xenografts. Methods Human endometrial carcinoma Ishikawa cells were cultured in vitro. The effects of PD98059 and LY294002 on proliferation, apoptosis, and cell cycle distribution of endometrial cancer cells were detected by monotetrazolium ( MTT) assay and fluorescence-activated cell sorting technique. The models of xenografted tumor were established by the subcutaneous inoculation in 24 nude mice, and then they were randomly divided into 4 groups ( n = 6) , normal saline group, PD98059 group (PD group) , LY294002 group ( LY group) or PD98059 + LY294002 group ( PD + LY group) by intraperitoneal injections, respectively. The anti-tumor efficacy was evaluated by measuring tumor volume and tumor growth status. The histopathological change of tumor specimens was observed using HE staining and terminal deoxynucleotidyl transferasemediated dUTP-digoxigen in nick and labeling method (TUNEL) testing and the expression levels of p-ERK and p-Akt were detected by immunohistochemistry method. Results ( 1) The proliferation of Ishikawa cells were suppressed after treated by PD98059 and ( or) Y294002, in which A570 values of cells decreased showing both time-dependent and concentration-dependent manner ( LY294002: Fgroup = 9. 801, P = 0. 002; Ftime = 10. 398, P = 0. 001. PD98059: Fgroup= 8. 213, P = 0. 015; Ftime = 6. 839, P = 0. 036). Cell cycle distribution analysis revealed that percentage of Ishikawa cells at G0/G1 phase(Ftime =35.049, P= 0.004; Fgroup = 32. 024, P ＜0. 01) increased and percentage of S phase cells (Ftime = 7. 789, P = 0. 049; Fgroup = 30. 132, P ＜0. 01) decreased significantly. The percentage of apoptotic cells increased significantly among PD group, LY group and PD + LY group, in which there were significant difference [(63. 3 ±0.5)% vs (30. 7 ± 20. 1) % vs(40. 8 ± 1. 3) % ; F = 621. 059, P ＜ 0. 01]. (2) Compared with the control group, the increasing of transplanting tumor volume in the treated groups were obviously ( F = 23. 545 , P ＜ 0. 01) , and the inhibited rate of the tumor was higher in PD + LY group than that in PD group or LY group [(68 ± 9 ) % vs ( 32 ± 16 ) % or ( 38 ± 17 ) % ; F = 10. 283 , P ＜ 0. 05]. ( 3 ) HE staining shown that there were different degrees of necrosis for endometrial carcinoma cell in different groups. The apoptosis of tumor cells were significantly increased in treated groups by TUNEL testing [(13. 7 ± 1. 5)% , ( 14. 1 ± 1. 2)% , (29. 0 ± 1. 8 ) % ; F = 320. 344, P ＜ 0. 01]. Immunohistochemistry results demonstrated that the expressions of p-ERK and p-Akt in treated groups were lower than that in control group, of which LY + PD group was the lowest one. Conclusion The signal pathway inhibitors PD98059 and LY294002 could inhibit the growth of human endometrial carcinoma in vivo and in vitro, in which may induce cell apoptosis.
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Objective To study the influence of survivin mutant-T34A ( survivinT34A) and survivin deletant-N-terminal 8 amino acids residues ( survivinN-8AA ) on the cell cycle distribution and chemosensitivity in human ovarian cancer HO-8910 cells for explorating the roles of modified survivin-mediated apoptosis induced by chemotherapeutic agents and possible signaling pathways involved. Methods pcDNA3.1 plasmid contained wild-type, survivinT34A and survivinN-8AA genes were transfected into HO-8910 cells,respectively, the control groups were HO-8910 cells transfected with pcDNA3. 1 plasmids. The expression of mRNA was examined by reverse transcription(RT) PCR and identified by DNA sequencing; the cell cycles were determined by flow cytometer analysis ( FCM ); the growth inhibitions rate of cisplatin ( DDP),paclitaxel (PTX) and LY294002 on the transfected cells were determined using methyl thiazolyl tetrazolium (MTT) assay. Results (1) The RT-PCR procedures and genome sequences showed that the survivin mRNA were expressed stable in the transfected HO-8910 cells. (2) There was lower percent of G0/G1 phase cells in SN-HO-8910 cells than that in PC-HO-8910 cells (44. 72% vs. 49.64%, P ＜0. 05) ;while higher percentage of G2/M phase and S phase cells( 1.06% and 54. 22% vs. 0. 56% and 49. 80%, P ＜ 0. 05 ).There was lower the G2/M phase and S phase cells in M-HO-8910 cells 0. 16% and 36. 33%, than that in PC-HO-8910 cells( P ＜ 0. 05 ); while higher percentage of G0/G1 phase cells(63. 51% ,P ＜ 0. 05 ). G0/G1 ,G2/M and S phase cells in Sur-HO-8910 cells were 54. 46%, 0. 62% and 44. 92%, and there were not significantly difference ( P ＞ 0. 05 ), compared to those in PC-HO-8910 cells. ( 3 ) The inhibitory concentration ( IC50 ) of DDP and PTX were higher in Sur-HO-8910 cells than those in control cells [(20. 4 ±6. 1)vs. (14.4 ±3.9)μmol/L,(36.7 ±4.0) vs. (28.6 ±3.6) μmol/L;all P＜0.05]. The IC50 of DDP and LY294002 in SN-HO-8910 cells were lower than those in control cells[(7. 6 ± 1.0) vs. ( 14. 4 ± 3.9)μmol/L, ( 13.2 ± 4. 0) vs. (41.0 ± 7. 9 ) μmol/L; all P ＜ 0. 01]. The IC50 of PTX [( 37. 9 ± 4. 8 ) μmol/L]in SN-HO-8910 cells were higher than that in control cells(P ＜0. 05). The IC50 of DDP in M-HO-8910 cells [(9.9 ± 1.2) μmol/L] were lower than that in control cells(P ＜0. 05) ,and the IC50 of LY294002 in M-HO-8910 cells [(66. 9 ± 4. 8) μ mol/L] higher than that in control cells ( P ＜ 0. 01 ). Conclusions The changes of cells cycle distribution caused by survivinT34A or survivinN-8AA enhanced the G2/M cell cycle-dependent chemosensitivity of PTX. Compared to survivinT34A, survivinN-8AA preferentially to mediate the cytotoxicity of DDP and LY294002, suggesting that it may be related to the cell cycle-dependence of survivin function and to blockage of the formation of its active dimer.
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Objective To explore the expression and significance of phosphatidylinositol-3 kinase (PI3K)in ovarian cancer,and the effect of combined PI3K inhibitor(LY294002)therapy with cisplatin on epithelial ovarian carcinoma,and to explore if there is a synergistic effect between the two therapies. Methods The expression levels of PI3K p85 subunit proteins and mRNA were evaluated by western blot and RT-PCR in normal ovarian tissue(G1),ovarian benign tumor tissue(G2),ovarian borderline tumor tissue(G3)and ovarian cancer tissue(G4),and the relevant clinical pathological parameters were analyzed.SKOV3 ceils were isolated and cultured by enzymolysis method.SKOV3 cells were treated with culture medium only,LY294002(1,10,30,50,100 ?mol/L),cisplatin(0.33,1.25,2.5,5, 10 ?mol/L),LY294002(50 ?mol/L)+cisplatin(10 ?mol/L)for 2 days,respectively.The effect of LY294002 and cisplatin on the growth of SKOV3 cells was measured by methyl thiazolyl tetrazolium assay. Results There was no positive expression of PI3K p85 subunit proteins in G1 and G2,while the expression was 2/6 in G3,and 85%(33/39)in G4.PI3K p85 subunit mRNA expression levels were 0.178?0.102 in G1,0.643?0.112 in G2,0.847?0.058 in G3,1.689?0.423 in G4;there was a significant difference between G1,G2,G3 and G4(P0.05).Significant differences were noted between protein expression levels in G4(Ⅲ,Ⅳ)and G4(Ⅰ,Ⅱ;P