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OBJECTIVE: To study the effect of isoalantolactone on inhibiting proliferation of MCF-7 and induce apoptosis in vitro and further to explore its machinism via mitochondrial and phosphatidylinositol 3-kinase/Akt signaling pathways. METHODS: The subject investigated isoalantolactone in MCF-7 cells proliferation inhibition by MTT and SRB methods, and matching the results of two methods; observing morphological changes by inverted microscope and each phase of apoptosis of MCF-7 cells effected by isoalantolactone after 24 h with Hoechst 33258 staining method. Using transmission electron microscopy to observe MCF-7 cells morphology change to determine the mechanism of research; rhodamine 123 tag, laser confocal scanning microscope detection isoalantolactone on the effect of MCF-7 cells mitochondrial membrane potential; Using Western blot to the expression of Bcl-2, Bax, Akt and p-Akt; detecting caspase-3 activity in MCF-7 cells by colorimetry method. RESULTS: Isoalantolactone has strong inhibition of proliferation in MCF-7 cells, IC50 values of MTT and SRB methods were 15.21 and 14.908 μg•mL-1, and in a dose -dependent manner; the morphological of MCF-7 cells was changed after the treatment by Hoechst staining method; morphological changes were observed under transmission electron microscopy of cells display, the drug group cells showed typical apoptotic characteristics of different periods. With the concentrations of isoalantolactone increasing, the level of mitochondrial membrane potential reduced. Western blot result showed that, isoalantolactone could down regulate the expression of anti apoptotic protein Bcl-2, p-Akt, and up regulate the expression of pro-apoptotic protein Bax, had no effect on the expression of Akt protein. And the activity of caspase-3 could be raised by increasing dosage, compared with the control group with significant difference(P < 0.01). CONCLUSION: Isoalantolactone effectively inhibites the proliferation of MCF-7 cells through mitochondrial and phosphatidylinositol 3-kinase/Akt signaling pathways, which is regulated by activation of caspase-3, down-regulation of Bcl-2, and up-regulation of Bax. Isoalantolactone-induced apoptosis is involved in mitochondrial and the PI3K/Akt pathway.
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Objective:To establish an HPLC gradient elution method for the determination of isoalantolactone,alantolactone,xan-thotoxol,oxypeucedanin,imperatorin and osthole in Kangfu ointment simultaneously. Methods:Isoalantolactone,alantolactone,xan-thotoxol,oxypeucedanin,imperatorin and osthole were determined by HPLC with a chromatographic column of Agilent TC-C18(250 mm ×4.6 mm,5 μm),the mobile phase was methanol-0.1% formic acid solution with gradient elution at a flow rate of 0.9 ml·min-1. The detection wavelengths were 220 nm and 300 nm. The column temperature was 35℃. Results: The linear range of isoalantolac-tone,alantolactone,xanthotoxol,oxypeucedanin,imperatorin and osthole was 6.16-123.20 μg·ml-1,3.78-75.60 μg·ml-1,1.87-37.40 μg·ml-1,4.06-81.20 μg·ml-1,9.27-185.40 μg·ml-1and 13.89-277.80 μg·ml-1,respectively. The correlation coef-ficient was 0.999 4,0.999 8,0.999 6,0.999 5,0.999 9 and 0.999 7, respectively. The average recovery was 98.04% (RSD=1.06%),97.10%(RSD = 1.53%), 96.73% (RSD = 0.90%), 98.92% (RSD = 1.36%), 99.12% (RSD = 0.83%) and 100.27%(RSD=0.58%),respectively. Conclusion:The method is simple and accurate,which can be used to improve the quality standard for Kangfu ointment.
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Objective To study the major chemical components from Inula helenium. Methods The compounds were separated and purifid by using a variety of chromatographic techniques including silica gel column chromatography, thin layer chromatography, and high performance liquid chromatography, and the structures of the compounds were verified by nuclear magnetic spectroscopy and literature data. Results A total of 22 compounds were separated from petroleum ether extract of I. helenium and identified separately as alantolactone (1), isoalantolactone (2), 4,4-dimethylsterols (3), 11αH,13-dihydroisoalantolactone (4), 11αH,13-dihydroalantolactone (5), 4(15)-epoxy-isoalantolactone (6), 5α,6α-epoxyalantolactone (7), alloalantolactone (8), isoalloalantolactone (9), friedelin (10), friedelinol (11), erythrodiol (12), β-sitosterol-glucopyranoside (13), lupeol acetate (14), lupeone (15), lupeol (16), δ-amyrin (17), lupeol palitate (18), 5,7,4'-trihydroxy-3',5'-dimethoxy flavane (19), (+)-syringaresinol (20), 3,5,3'-trihydroxy-6,7,4'-trimethoxy flavone (21), and 3,5,6,7,3'-hydroxy-4'-methoxy dihydroflavones (22). Conclusion Compounds 21 and 22 are isolated from this genus for the first time; Compounds 10-15, 17, and 18 are separated from the I. helenium for the first time. After antibacterial test, compounds 1, 2, 4, 5, 7-9 have strong inhibitory effects on Escherichia coli, Staphylococcus aureus, and Bacillus subtilis.
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OBJECTIVE:To establish the method for simultaneous determination of alantolactone,isoalantolactone,gallic acid,emodin,aloe-emodine,rhein,physcion and chrysophanol in Liuwei nengxiao pills.METHODS:HPLC method was adopted.The determination was performed on Diamonsil C18 with mobile phase consisted of methanol-acetonitrile-0.1％ glacial acetic acid (gradient elution) at the flow rate of 1.0 mL/min.The detection wavelengths were set at 254 nm (alantolactone,isoalantolactone,emodin,aloe-emodine,rhein,physcion and chrysophanol),270 nm (gallic acid).The column temperature was 25 ℃,and sample size was 10 μ L.RESULTS:The linear ranges of alantolactone,isoalantolactone,gallic acid,emodin,aloe-emodine,rhein,physcion,chrysosphanol were 0.121-3.63 μg(r=0.999 9),0.122-3.66 μg(r=0.999 9),0.219-6.57 μg(r=0.999 9),0.016 4-0.492 μg(r=0.999 7),0.017 3-0.519 μg(r=0.999 9),0.015 3-0.459 μg(r=0.999 9),0.007 2-0.216 μg(r=0.999 9),0.016 2-0.486(r=0.999 9).The limits of quantification were 0.41,0.26,0.35,0.13,0.17,0.14,0.15,0.13 ng;limits of detection were 0.12,0.08,0.11,0.04,0.05,0.04,0.05,0.04 ng.RSDs of precision,stability and reproducibility tests were all lower than 2.0％.The recoveries were 98.05％-102.46％ (RSD=1.75 ％,n=6),98.55％-102.89％ (RSD=1.91％,n=6),98.53 ％-102.34％ (RSD=1.66％,n=6),101.71％-103.41％ (RSD =0.57 ％,n=6),101.04％-103.01％ (RSD=0.69％,n=6),101.63％-102.75％ (RSD=0.39 ％,n=6),96.94％-101.11％ (RSD=1.61％,n=6),98.06％-99.10％ (RSD=0.40％,n=6).CONCLUSIONS:The method is accurate,simple and suitable for simultaneous determination of 8 components in Liuwei nengxiao pills.
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Objective To explore the effects of isoalantolactone on the proliferation of human chronic myelogenous leukemia drug-resistant cell line K562/A02. Methods K562/A02 cells were treated with 6.25, 12.5, 25, 50 and 100 μmol/L isoalantolactone for 24 and 48 h, cell viability was analyzed with MMT assay. K562/A02 cells were treated with 10, 15, 20 μmol/L isoalantolactone for 24 h. Flow cytometry was used to examine the effect of isoalantolactone on the cell-cycle and apoptosis of K562/A02 cells. The related proteins were analyzed using Western blot. One-way ANOVA was used for statistical analysis. Results Isoalantolactone effectively inhibited the proliferation of K562/A02 cells in a dose-dependent manner (P<0.05) with IC50 value of (15.00 ±1.03) μmol/L at 24 h, respectively; Flow cytometry displayed that isoalantolactone may induce K562/A02 cells apoptosis in a concentration-dependent manner (P<0.05). The apoptotic rate significantly increased from (2.71 ±0.52) % in the control group to (19.10 ±1.55) %, (27.61 ± 2.32)%and (32.01±3.01)%(F=33.901, P<0.05), respectively, after treatment with 10, 15, and 20 μmol/L of isoalantolactone for 24 h. The percentage of cells in the S phase increased from (57.80±2.11) % to (68.62± 2.89)%, (78.41±3.51)%and (80.61±2.90)%, respectively (F=51.328, P<0.05). Western blot indicated that the expression of bcl-2, p-bcr-abl, p-STAT5, CDK2 and cyclin A significantly decreased (P< 0.05), and that of cytochrome C, Bax, and p21 increased with the increasing of isoalantolactone concentration (P< 0.05). Conclusion Isoalantolactone can significantly inhibit the proliferation of K562/A02 cells through bcr-abl-STAT5 signaling pathway.
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Objective:To investigate the induction of apoptosis by isoalantolactone in human cervical cancer Hela cells is mediated through ROS generation and Mitochondrial dysfunction. Methods: Cells were treated with isoalantolactone in a dose-dependent manner in the presence or absence of NAC for 24 h as the experimental group,and the normal cells were used as control group. Cell viabilities were determined by the MTT assay;the nuclear morphology of Hela cells were observed under fluorescence microscope using the Hoechst 33258 staining;apoptosiscell cycle and reactive oxygen species ( ROS ) and mitochondrial membrane potential(MMP) were measured by flow cytometry;the protein expression levels of cytochrome C,Bcl-2,Bax and Caspase-3 were detected by Western blot. Results:In the present study,we found that isoalantolactone inhibits growth in a dose-dependent manner in Hela cells. Further studies revealed that Hela cells were treated with 20 and 40 μmol/L isoalantolactone for 24 h,after which we could observe the fragmented nuclei and the increased apoptosis rate. And we also found that isoalantolactone arrested the cell cycle at S phase and increased generation of reactive oxygen species and dissipation of mitochondrial membrane potential (△ψm) in Hela cells. While pretreatment with NAC obviously blocked the apoptotic and inhibition effect of isoalantolactone indicating that induction of apoptosis is ROS-dependent,Western blot study showed that isoalantolactone increased the expression of Bax and cleaved Caspase-3 and decreased the expression of Bcl-2 with concomitant release of cytochrome C from mitochondria into cytosol. Conclusion: Isoalantolactone could inhibit the proliferation and induce the apoptosis of human cervical cancer Hela cells in vitro through mediating ROS generation and Mi-tochondrial dysfunction, the mechanism of which is also accompanied by up-regulation of Bax expression, down-regulation of Bcl-2 expression,activation of Caspase-3 and release of cytochrome C.
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Objective To establish a method to simultaneously determine contents of alantolactone and isoalantolactone in Sanwei Ganlusan. Methods Contents of alantolactone and isoalantolactone was determined by HPLC was used with PDAD detector;the column was CAPCELL PAK MG C18 (4.6 mm×250 mm, 5μm);the mobile phase consisted of acetonitrile-0.4%phosphoric (58∶42);the flow rate was 1.0 mL/min;the detection wavelength was set at 225 nm;the column temperature was maintained at 30 ℃. Results The linear range of alantolactone was 0.083-0.517 μg (r=0.999 9), and isoalantolactone was 0.108-0.672 μg (r=0.999 9). The mean recovery of alantolactone was 97.95%, RSD=1.31%. The mean recovery of isoalantolactone was 97.69%, RSD=1.24%. Conclusion The method is accurate and simple in operation, which can be used to simultaneously determine contents of alantolactone and isoalantolactone in Sanwei Ganlusan.
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Abstract: In this article, we report a study of assay of sesquiterpene lactones (alantolactone, isoalantolactone) in plant extraction derived by ultrasound-assisted extraction, оrthogonal test design and reflux extraction from medicinal plant’s composition (Salsola laricifolia turcz.e litv+Inula helenium). High-performance liquid chromatography (HPLC) method was used for determination of the contents of alantolactone and isoalantolactone in the investigated extracts. The result shown that the amount of alantolactone was 0.64±0.03%, and isoalantolactone 0.59±0.01% in the plant extraction derived by reflux condensation extraction. Key words: Alantolactone, Isoalantolactone, HPLC, Salsola laricifolia turcz.ex litv, Inula helenium, Reflux method, Ultrasound- assisted extraction.
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Objective To establish the quality standard for Tibetan medicine MNXT granule.Methods Inula racemosa,Tinospora sinensis were identified by TLC; Isoalantolactone and alantolactone were determined by HPLC.Results Inula racemosa,Tinospora sinensis could be identified by TLC.The concentration of isoalantolactone was linear in the range of 0.281～0.842 μg,r=0.9998,with the average recovery rate being 98.5％,RSD being1.14％.The concentration of alantolactone was linear in the range of 0.232～0.696 μg,r=0.9999,with the average recovery rate being 97.4％,RSD being 1.10％.Conclusion The method was simple,accurate,repeatable and able to control the quality of preparation effectively.