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Objective:To explore the synergistic effect of arbuscular mycorrhizal（AM） fungi mixed inoculation on the growth，physiological and biochemical characteristics，root biomass and terpenoid component accumulation of Aucklandia lappa seedlings，so as to provide a reference for the combination and application of the dominant complementary effect mycorrhizal fungi. Method:The effect of different AM fungi combined with inoculation on the root mycorrhizal infection rate，plant growth，physiological and biochemical characteristics，root biomass，costunolide and dehydrocostus lactone of A. lappa seedlings were determined by pot inoculation at room temperature. Result:It was found that AM fungi could form good mycorrhizal symbiosis with the roots of A. lappa.The formation of mycorrhizal symbiosis system could increase the chlorophyll content of A. lappa leaves，increase the activities of catalase（CAT），peroxidase （POD），superoxide dismutase （SOD），reduce the content of malondialdehyde（MDA），and promote photosynthesis of A. lappa. Compared with CK group，AM fungus treatment could significantly promote the accumulation of costunolide and dehydrocostus lactone，and the accumulation of its metabolites，costunolide and dehydrocostus lactone，into roots during the symbiotic cultivation of A. lappa seedlings，indirectly improving the quality of medicines and yield of alantolactone. Conclusion:Inoculation of AM fungi can improve the root mycorrhizal viability，increase the absorption of nutrients and promote the growth of woody incense.The mixed inoculation treatment of S2，S4 and S5 had the best mycorrhizal effect in artificial cultivation，and the growth and medicinal quality of A. lappa were the best，which provided technical support for the application and popularization of A. lappa mycorrhizal biotechnology.
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Objective: To establish a quantitative analysis of multi-components by single marker (QAMS) method for the simultaneous determination of six components of gallic acid, hydroxysafflor yellow A, geniposide, ellagic acid, costunolide and dehydrocostus lactone in Gurigumu-13 Pill, which is proved to be a scientific and feasible method in the quality analysis in Gurigumu-13 Pill. Methods: The relative factor (fs/i) of gallic acid, ellagic acid, hydroxysafflor yellow A, costunolide and dehydrocostus lactone were established by HPLC method with geniposide as internal standard, which were used to calculate the content of five constituents in the samples of Gurigumu-13 Pill. Meanwhile, external standard method (ESM) was used to calculate the content of six constituents. The difference between QAMS and ESM was analyzed to evaluate the accuracy of QAMS. Results: The fs/i of gallic acid, hydroxysafflor yellow A, ellagic acid, costunolide and dehydrocostus lactone were 0.481 0, 0.906 4, 0.170 9, 0.971 2 and 1.261 5, respectively. The content determination results of six batches of Gurigumu-13 Pill were calculated by the method of QAMS and ESM, with no significant difference in RSD < 2.0%. Conclusion: The fs/i established in the QAMS method with geniposide as the internal reference substance is accurate and simple. The QAMS method can be used for the multi-index quality evaluation of Gurigumu-13 Pill.
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OBJECTIVE： To establish a method for simultaneous determination of matrine， oxymatrine， gallic acid， peoniflorin， costunolide and dehydrocostus lactone in Libiling tablets. METHODS： RP-HPLC method was adopted. The determination was performed on Agilent ZORBAX SB-C18 column with mobile phase consisted of methanol-0.1％ phosphoric acid （gradient elution） at the flow rate of 1.0 mL/min. The detection wavelengths were 210 nm （matrine， oxymatrine） and 225 nm （gallic acid， peoniflorin， costunolide， dehydrocostus lactone）. The column temperature was set at 30 ℃， and sample size was 5 μL. RESULTS： The linear ranges of matrine， oxymatrine， gallic acid， peoniflorin， costunolide， dehydrocostus lactone were 0.053-5.28 mg/mL（r＝0.999 8）， 0.125-12.54 mg/mL（r＝0.999 9）， 0.013-1.33 mg/mL（r＝0.999 8）， 0.169-16.94 mg/mL（r＝0.999 9）， 0.048-4.77 mg/mL（r＝0.999 8）， 0.072-7.16 mg/mL （r＝0.999 9）. The limits of quantitation were 4.08×10－4， 4.48×10－4， 3.12×10－4， 2.10×10－4， 1.36×10－4， 1.84×10－4 mg/mL， respectively. The limits of detection were 1.24×10－4， 1.50×10－4， 1.02×10－4， 6.20×10－5， 4.20×10－5， 6.40×10－5 mg/mL， respectively. RSDs of precision， stability and reproducibility tests were all lower than 2％ （n＝6）. The recoveries were 98.03％-101.43％ （RSD＝1.25％， n＝6）， 97.73％-102.26％ （RSD＝1.96％， n＝6）， 97.18％-101.41％ （RSD＝1.98％，n＝6）， 97.45％-102.11％ （RSD＝1.88％，n＝6）， 96.85％-101.07％ （RSD＝1.75％， n＝6）， 97.12％-102.64％ （RSD＝1.82％，n＝6）， respectively. CONCLUSIONS： Established method is simple， stable and rapid， and can be used for simultaneous determination of 6 components in Libiling tablets.
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Objective To explore the effect of dehydrocostus lactone on the proliferation of chronic myeloid leukemia cell line K562 and its mechanism.Methods K562 cells in logarithmic growth phase were treated with different concentrations of dehydrocostus lactone.Cell morphology was observed by Wright-Giemsa staining.Cell proliferation was detected by CCK-8 method.Cell cycle,apoptosis and the expressions of cell surface differentiation antigen CD14 and CD1 1b were detected by flow cytometry.JAK-STAT pathway and the expressions of apoptosis and cell cycle related proteins were detected by Western blot.Results Compared with the control group,the proliferation of K562 cells could be inhibited after treatment with different concentrations (4.0,6.0,8.0,10.0 and 12.0 μmol/L) of dehydrocostus lactone for 24 h,and the difference was statistically significant (F =109.510,P ＜ 0.05).After treatment with 5.0 and 10.0 μmol/L dehydrocostus lactone for 24 h,the apoptosis rates of K562 cells were (16.1±3.8)％ and (29.6±4.3)％,which were higher than that of the control group [(3.1±0.5)％] (F =83.255,P ＜ 0.05).After treatment with 5.0 and 10.0 μmol/L dehydrocostus lactone for 24 h,the proportion of G2/M phase cells in K562 cells were (17.0±3.2)％ and (28.8± 3.9)％,which were higher than that of the control group [(9.1±2.3)％] (F =161.598,P ＜ 0.05);the proportion of S phase cells in K562 cells were (48.1±3.9)％ and (61.0±5.4)％,which were higher than that of the control group [(39.6±3.6)％] (F =192.356,P ＜ 0.05).After treatment with 2.5 and 5.0 μmol/L dehydrocostus lactone for 72 h,the expression rates of CD14 in K562 cells were (28.6±3.9)％ and (41.1±4.4)％,which were higher than that in the control group [(3.1±0.5)％] (F =132.811,P ＜ 0.05);the expression rates of CD11b in K562 cells were (42.4±5.0)％ and (61.2±5.7)％,which were higher than that in the control group [(4.2±1.1)％] (F =179.553,P ＜ 0.05).Dehydrocostus lactone could decrease the expressions of JAK2,STAT5,eyclin E,CDK2,cyclin A,CDC25C,cyclin B1,CDK1 and bcl-2 proteins,and up-regulate the expressions of p21 and bax proteins.Conclusion Dehydrocostus lactone can inhibit the proliferation of chronic myeloid leukemia K562 cells,which may be achieved by cell cycle arrest,induction of apoptosis and differentiation.
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Objective: To prepare pegylated long-circulating liposomes co-encapsulated by costunolide (Cos) and dehydrocostus lactone (DL), optimize the formulation and process, and evaluate the quality. Methods: The pegylated long-circulating liposomes co-encapsulated by Cos and DL were prepared by film hydration method. Single factor test and Box-Behnken response surface methodology were used to optimize the preparation process with encapsulation efficiency of Cos and DL as the index. The particle size, surface potential, encapsulation efficiency and in vitro release of the liposomes were evaluated. Results: The optimal preparation conditions were as follows: drug-to-lipid ratio was 0.14, ratio of cholesterol to phospholipid was 0.05, mPEG-2000-DSPE addition amount was 6%, hydration time was 30 min, and probe ultrasonic time was 4 min. The obtained liposome was round and uniform in distribution, with an average particle size of (104.8 ± 2.48) nm, a polydispersity index (PDI) of (0.245 ± 0.031), and a Zeta potential of (-9.7 ± 0.23) mV, the encapsulation efficiency of Cos and DL were (91.9 ± 2.6)% and (94.41 ± 1.23)%, respectively. Conclusion: The PEGylated long-circulating liposome prepared by the process and prescription optimization has good appearance and high encapsulation efficiency, which can meet the application requirements.
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Objective:To establish a supercritical fluid chromatography(SFC) method for separating and purifying costunolide and dehydrocostus lactone in Aucklandiae Radix. Method:With supercritical carbon dioxide as the mobile phase,the effect of six factors, such as type of chromatographic columns,modifiers and modifiers ratio, flow rate of mobile phase,pressure and temperature, on the separation process of supercritical fluid chromatography were explored. The target components were separated and prepared by semi-preparative supercritical fluid chromatography. High performance liquid chromatography and nuclear magnetic resonance were used to analyze the components and study the thermodynamic regularity of the chromatographic process. Result:C18 column (10 mm×250 mm,5 μm) was adopted, with supercritical fluid dioxide as the mobile phase,the ratio of methanol was 0.13%,the flow rate was 12 mL·min-1,column pressure was 13 MPa,column temperature was 318℃, and detection wavelength was 225 nm. The sample was injected for 20 times,crude extract was 4 mg,and each target component was collected according to the chromatogram. Its purity was determined to be more than 99%by HPLC,and its structure was determined as costunolide and dehydrocostus lactone by NMR. Under this condition,the SFC separation process was normal-phase chromatography. Conclusion:The method can be used to prepare effective components of Aucklandiae Radix with a high purity and low solvent residue.
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Introduction@#After central nervous system injury, microglia cells are activated to initiate inflammatory responses and release cytokines that beneficially or detrimentally affect surrounding cells. Lipopolysaccharide stimulates microglia cells and produce pro-inflammatory cytokines such as interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-α. A dehydrocostus lactone (DDL) which is contained in medicinal plant, Saussurea lappa, is considered to have various health benefits in neurodegenerative diseases of central nervous system. </br> In this study, we aimed to investigate the anti-inflammatory effects of Dehydrocostus Lactone following lipopolysaccharide stimulation of microglial cells in vitro.@*Materials and Method@#The anti-inflammatory effects of dehydrocostus lactone were studied using lipopolysaccharide (LPS) stimulated murine microglia (BV2). BV2 were cultered in DMEM then three different doses (4µM, 8µM and 12µM) of DDL were added in the medium for 30 minutes respectively. Then BV2 were treated with 1 ng/ ml LPS for 24 hours to stimulate. The level of IL-1β, IL-6 and TNF-α were measured in 100µl of culturemedium supernatant by ELISA. Three different doses of DDL anti-inflammation groups (BV2+DDL+LPS), LPS-activated group (BV2+LPS) and control group (only BV2) were analysed. @*Results@#LPS-treated BV2 cells had increased IL-1β, IL-6 and TNF-α compared with those without LPS treatment. Pretreatment with dehydrocostus lactone prior to LPS treatment significantly decreased levels of IL-1β and TNF-α in a dose-dependent manner compared with LPS-treated BV2 cells and 4µM was the most effective anti-inflammatory dose of dehydrocostus lactone. As for IL-6, 12µM dehydrocostus lactone was the most effective anti-inflammatory dose, although all doses significantly decreased the level of IL-6, in a dose-dependent manner. @*Conclusion@#These results show that DDL decrease inflammation related IL-1β, IL-6 and TNF-α in a dose-dependent manner in microglia cells.
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Objective:To establish a GC method for the imultaneous determination of cinnamaldehyde, bornyl acetate, costunol-ide,dehydrocostus lactone,magnolol and honokiol in Dutong pills. Methods: The determination was performed on an HP-5 column (30 m ×0.32 mm,0.25 μm) with programmed temperature. The carrier gas was nitrogen with the flow rate of 2.0 ml·min-1. The injection volumn was 1 μl and the sample split ratio was 5:1. The inlet temperature was 280 ℃. The detector was a flame ionization detector with temperature at 300 ℃. Results:The linear ranges were 32.28-516.40 μg·ml-1for cinnamaldehyde(r=0.999 3), 27.06-433.00 μg·ml-1for bornyl acetate(r=0.999 2),25.65-410.40 μg·ml-1for costunolide(r=0.999 3),26.10-417.60 μg ·ml-1for dehydrocostus lactone(r=0.999 3),24.01-384.20 μg·ml-1for magnolol(r=0.999 0) and 18.32-293.10 μg·ml-1 for honokiol(r=0.999 4). The average recovery was 99.71%(RSD=0.67%),99.34%(RSD=1.18%),100.16%(RSD=0. 34%),100.40%(RSD=0.39%),99.32%(RSD=1.22%) and 99.58%(RSD=0.58%)(n=6),respectively. Conclusion:The method is simple,sensitive and accurate,can be used to supplement the insufficient quality control of Dutong pills.
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Objective To develop an HPLC wavelength switching combined gradient elution method for simultaneous determi?nation of nine components in Kanglixin Jiaonang(KLXJN). Methods The analysis was performed on a Kromasil C18(4.6 mm×250 mm, 5μm)with gradient elution by using the mobile phase of acetonitrile-methanol(1:2)(A)-0.1%phosphoric acid solution(B). The col?umn temperature was maintained at 30℃and the flow rate was 0.9 ml/min. The detection wavelengths were set at 225 nm for costuno?lide(1)and dehydrocostus lactone(2),254 nm for aloe-emodin(3),rhein(4),emodin(5)and physcion(6),and 425 nm for bisde?methoxycurcumin(7),demethoxycurcumin(8)and curcumin(9). Results The calibration curves were linear within the range(μg/ml)of 6.610-132.2(r=0.9999)for 1,7.890-157.8(r=0.9996)for 2,14.07-281.4(r=0.9992)for 3,3.450-69.00(r=0.9997)for 4, 2.670-53.40(r=0.9998)for 5,3.760-75.20(r=0.9999)for 6,5.880-117.6(r=0.9996)for 7,8.490-169.8(r=0.9993)for 8,and 13.91-278.2(r=0.9991)for 9,respectively. The recoveries for 1,2,3,4,5,6,7,8 and 9 were 98.28%(RSD=1.09%),97.76%(RSD=0.80%),99.08%(RSD=1.72%),97.19%(RSD=1.00%),98.45%(RSD=1.24%),96.96%(RSD=1.21%),98.51%(RSD=1.55%), 97.52%(RSD=0.83%),and 100.04%(RSD=0.93%),respectively. Conclusion The established method is accurate,rapid and can be used for the quality control of KLXJN.
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OBJECTIVE:To establish a method for the simultaneous determination of costunolide and dehydrocostus lactone in Haoweilai soft capsule. METHODS:HPLC was performed on the column of Dikma C18 with mobile phase of acetonitrile- water (V/V,55∶45) at a flow rate of 1.0 ml/min,detection wavelength was 225 nm,column temperature was 25 ℃,and the injection volume was 10 μl. RESULTS:The linear range were 24.00-108.00 μg/ml(r=0.999 7) for costunolide and 20.88-93.98 μg/ml for dehydrocostus lactone (r=0.999 8);RSDs of precision,stability and reproducibility tests were lower than 1%;recoveries were 98.71%-100.00%(RSD=0.25%,n=6)and 96.88%-99.18%(RSD=0.40%,n=6). CONCLUSIONS:The method is simple with good stability and reproducibility,and can be used for the simultaneous determination of costunolide and dehydrocostus lactone in Haoweilai soft capsule.
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Objective To establish the method to analyze the contents of Costunolide and Dehydrocostus Lactone in Tibetan medicineLiuwei Muxiang Pill.Methods HPLC method was used; the XDB-C18 column (4.6 mm × 50 mm, 1.8μm) was adopted;the mobile phase was methanol- water (60∶40);the flow rate was 0.8 mL/min;UV detection was 225 nm.Results The linear range of Costunolide and Dehydrocostus Lactone was 0.235 5-1.413μg and 0.291 3-1.748μg. The average recoveries were 99.25% and 99.41%, respectively.Conclusion The method is accurate, reliable, stable and with good repeatability, which provides the basis for the quality evaluation of Tibetan medicineLiuwei Muxiang Pill.
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Objective:To develop an HPLC method for the simultaneous determination of paeoniflorin, liquiritin, magnolol, ferulic acid, hesperidin, costunolide, dehydrocostus lactone, synephrine, baicalin, and berberine in Xianglian Huazhi Pill. Methods: The chromatographic separation was achieved on a Waters Symmetry-C18 (150 mm × 4.6 mm, 3.5 μm) column with methanol- acetonitrile-waters (50∶45∶5, A) and methanol-0.1% phosphoric acid (4∶96, B) as mobile phases for gradient elution, at the flow rate of 1.0 mL/min; The column temperature was 45 ℃. Results: The results showed that the ten active components were well separated and showed good linearity, paeoniflorin (1.5-15.0 μg/mL), liquiritin (0.5-5.0 μg/mL), magnolol (1.0-10.0 μg/mL), ferulic acid (0.5-5.0 μg/mL), hesperidin (12.5-125.0 μg/mL), costunolide (2.5-25.0 μg/mL), dehydrocostus lactone (2.5-25.0 μg/mL), synephrine (0.75-7.5 μg/mL), baicalin (1.5-15.0 μg/mL), and berberine (0.75-7.5 μg/mL). The precision was good and RSD was less than 2.0%. The repeatability was good, and RSD was less than 2.0%. The stability was good in 12 h. The average recoveries were between 98.5%-101.4%, and RSD was less than 2.0%. Conclusion: The method is accurate, sensitive, credible, and repeatable. It can be applied to the quality control of Xianglian Huazhi Pill.
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Objective: To compare the metabolic profiling of Vladimiriae Radix (VR) before and after simmer processing and to screen out the characteristic constituents leading to some differences. Methods: The chromatogram data sets of the processed and unprocessed VR were obtained by HPLC-UV analysis; The metabolic profiling differences of VR before and after processing were compared by principal component analysis, partial least square-discriminant analysis, and hierarchical cluster analysis, then the characteristic constituents were screened out. Results: The metabolic profilings between processed and unprocessed VR were apparently different, and the data showed seven constituents among them mainly made the quality difference, including costunolide and dehydrocostus lactone. Conclusion: The processed and unprocessed VR have the significant differences on the metabolic profiling.
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AIM:To study the difference of the chemical constituents between raw Radix Aucklandiae and Radix Aucklandiae roasted with wheat bran.METHODS:UV,HPLC and TLC were performed to analyze the chemical constituents before and after the processing.The constituents absorbed by wheat bran during the processing A.lappa.were isolated by silica gel CC.Their structures were identified by spectral analysis.RESULTS:The result of UV,HPLC and TLC showed that change of the chemical constituents in after the roasting had taken place.Two maior chemical constituents absorbed by wheat bran were dehydrocostus lactone and costunolide.CONCLUSION:The difference in sesquiterpeneoide before and after the processing of Radix Aucklandiae may be the evidence that the roasting with bran is to the benefit oftherapeutic effect.
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Objective To establish a determination method for dehydrocostus lactone in Dahuolu Pill.Methods TLC-Scanning was used.Results The average recovery was 98.30 %and RSD was 1.90 %.Conlusion The method is simple,accurate,repeatable,and can be used for the reference of the quality control to Dahuoluo Pill.
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AIM:To study the difference of the chemical constituents between raw Radix Aucklandiae and Radix Aucklandiae roasted with wheat bran.METHODS:UV,HPLC and TLC were performed to analyze the chemical constituents before and after the processing.The constituents absorbed by wheat bran during the processing A.lappa.were isolated by silica gel CC.Their structures were identified by spectral analysis.RESULTS:The result of UV,HPLC and TLC showed that change of the chemical constituents in after the roasting had taken place.Two major chemical constituents absorbed by wheat bran were dehydrocostus lactone and costunolide.CONCLUSION:The difference in sesquiterpeneoide before and after the processing of Radix Aucklandiae may be the evidence that the roasting with bran is to the benefit of therapeutic effect.