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Objective To establish the method for content determination of three lignans of Dendrobium Fimbriatum Hook..Methods The lignans in Dendrobium tasselii were identified by high-performance liquid chromatography/multi-stage mass spectrometry(HPLC-ESI/MSn)coupled with ultraviolet absorption spectrometry(UV)coupled with retention time localization of high-performance liquid chromatography(HPLC).The separation was carried out on a Kromasil 100-5 C18 column(4.6 mm×250 mm,5 μm)using a gradient elution of acetonitrile-0.1%formic acid solution as the mobile phase,the volume flow rate was 0.8 mL·min-1 and the column temperature was 35℃,and the mass spectrometry was performed using an ESI ion source with the data collected in the negative ion mode.The HPLC content was determined on the same column as that of MS analysis,with the mobile phase methanol + acetonitrile(V/V=1∶1)-0.01 mol/L ammonium acetate solution,gradient elution,flow rate of 0.8 mL·min-1,column temperature of 40℃,and detection wavelength of 215 nm.Results Syringaresinol di-O-glucoside and(-)-Syringaresinol 4-O-β-D-glucopyranoside and DL-Syringaresinol were identified from Dendrobium fimbriatum Hook.,and the results of content determination showed that the linear ranges of above three components were respectively 0.1701-3.4020,0.1020-2.0400,0.0403-0.8060 μg(r≥0.9995),the average recoveries were in the range of 97.71%-101.67%,and the relative standard deviations(RSDs)were all less than 3.0%.The contents of Syringaresinol di-O-glucoside and(-)-Syringaresinol 4-O-β-D-glucopyranoside and DL-Syringaresinol in the 10 batches of samples were 0.7779-1.3852,0.0734-0.1966,0.0295-0.1882 mg·g-1.Conclusion This research method can provide a reference basis for the quality evaluation method of Dendrobium fimbriatum Hook..
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Objective: To optimize the HPLC characteristic spectrum of flavonoid glycosides of Dendrobium officinale,and identify the common and specific components of different provenances. Method: Kromasil 100-5 C18 column was adopted, with tetrahydrofuran-acetonitrile-methanol (10:22:5)-0.05% phosphoric acid as mobile phase (gradient elution). The detection wavelength was 340 nm,the column temperature was 30℃,and the flow rate was 1.0 mL ·min-1. Result: 13 flavonoid characteristic peaks were marked in 27 batches of D. officinale,and 7 characteristic peaks of 6 flavonoid C-glycosides (vicenin Ⅱ,vicenin Ⅰ,schaftoside,isoschaftoside,violanthin and isoviolanthin) and one flavonoid O-glycosides (rutin) was identified. 7-11 characteristic peaks were detected in different batches of samples. Among them,vicenin Ⅱ was a relatively stable common peak in different source samples,and the characteristic peaks of rutin,schaftoside and isoschaftoside were quite different. According to the relative abundance of the characteristic peaks,the samples could be divided into three categories. Among them,the first category had 10 batches of samples,which mainly came from Danxia landforms of Guangdong,Jiangxi,Fujian and Zhejiang (Wuyi) Province (which called "Danxia landform species") and characterized by detection of obvious peak of rutin. The second category had 11 batches of samples,which mainly came from Yunnan and Guangxi Province (which included "Yunnan Guangnan species" and "Guangxi Tiepilan species") and characterized by detection of violanthin and isoviolanthin. And the third category had 6 batches of samples, which were mainly derived from Zhejiang Province (which called "native species from Zhejiang") and characterized by detection of different degrees of rutin peak, but it was difficult to detect the characteristic peaks of violanthin and isoviolanthin. HPLC characteristic chromatograms of D. officinale in bionics wild cultivation and greenhouse of "Danxia landform species" and "Guangxi Tiepilan species" were compared. The results showed that the characteristic peaks in D. officinale planted in greenhouse could be detected stably,which verified the reliability of the source in D. officinale. Conclusion: The analytical method has a better separation effect on flavonoids of D. officinale, with a good reproducibility. The commonness and specificities of flavonoid glycosides components of D. officinale from different categories have basically confirmed. This suggests that Vicenin Ⅱ is suitable to be a reference peak for characteristic chromatogram. Both the relative abundance of rutin and the detection or relative abundance of violanthin and isoviolanthin peaks could be used as a reference to judge the categories of D. officinale in "Danxia landform species" or "Tiepilan species from Yunnan, South Guangdong and Guangxi" or "native species from Zhejiang".
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Objective: To extract,isolate,purify and identify the structures of the flavonoid glycoside in Dendrobium officinale from two different origin places (Danxia species and Yunnan Guangnan species),and provide experimental reference for confirming the common flavonoid glycoside components in D. officinale. Method: ① 70% ethanol was applied to extract the total flavonoids in leaves of D. officinale from two different species. Organic solvents petroleum ether,acetic ether and water saturated n-butyl alcohol were used in turn to extract the crude extraction. Then AB-8 Macroporous resin,Sephadex LH-20 and ODS chromatographic column were applied to isolate and purify the water saturated n-butyl alcohol extraction fraction. The structures of flavonoid glycoside were identified by studying physicochemical property,applying modern spectroscopy method like HPLC,ESI-MSn,1H-NMR,13 C-NMR,etc. ② HPLC characteristic spectrum technique was used to analyse and compare the common flavonoid glycoside components in Dendrobium officinale from different origin places (Danxia species,Yunnan Guangnan species,Guangxi Tiepilan species and Zhejiang native species). Result: Five flavonoid glycoside compounds were isolated from the crude extractions of the leaves of D. officinale from two different species,and they were identified as rutin,vicenin Ⅱ,viceninⅠ,violanthin and isoviolanthin. The characteristic spectrum of vicenin Ⅱ and viceninⅠwere detected in stems of D. officinale from four different origin places (Danxia species,Yunnan Guangnan species,Guangxi Tiepilan species and Zhejiang native species),and vicenin Ⅱ had a better separation degree in the characteristic spectrum. However,the characteristic spectrum of violanthin and isoviolanthin were more obvious in Yunnan Guangnan species and Guangxi Tiepilan species,while rutin was obvious in the Danxia species. Conclusion: Vicenin Ⅱis the common flavonoid glycosides component in D. officinale from different origin places (Danxia species,Yunnan Guangnan species,Guangxi Tiepilan species and Zhejiang native species),and can be used as the internal reference material for the characteristic spectrum of D. officinale.
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Objective: To optimize the pre-column derivation high performance liquid chromatography (HPLC) content determination method of D-mannose and D-glucose as well as the content determination method of narinhenin in Dendrobium officinale and D. huoshanense, and compare the contents of D-mannose,D-glucose and narinhenin between D. officinale and D. huoshanense. Method: A pre-column derivation HPLC method modified by Chinese Pharmacopoeia(Ch.P) 2015 was used to simultaneously determine the contents of D-mannose and D-glucose,with acetonitrile-0.02 mol·L-1 ammonium acetate solution as mobile phase for gradient elution. Kromasil 100-5 C18 was performed with the wavelength set at 250 nm,and the flow rate was 1 mL·min-1;column temperature was 30℃. HPLC content determination of narinhenin was performed on Kromasil 100-5 C18 with the acetonitrile-methanol-0.4% phosphoric acid solution as mobile phase for gradient elution,and the wavelength was set at 290 nm; the flow rate was 0.8 mL·min-1,and column temperature was 40℃. Result: D-mannose and D-glucose showed a good linear relationship within the range of 0.15-3.0 μg and 0.075-2.25 μg (r=0.999 9); and their average recoveries were 99.01% (RSD 2.1%) and 101.69% (RSD 2.0%) respectively. In addition, the other methodological researches such as repeatability and durability all met the requirements. The contents of D-mannose(Cm),D-glucose(Cg) and sum of them (Cm+Cg) were 12.75%-36.40%,2.93%-18.39% and 19.23%-54.58% in 43 batch of D. officinale. Almost all of the results except very few samples reached the D-mannose standard in Ch.P 2015, and the total content of D-mannose and D-glucose was also up to the total polysccharide standard in Ch.P. The correlation between content and origin was not significant. The contents of D-mannose(Cm),D-glucose(Cg) and sum of them (Cm+Cg) were 14.33%-29.47%,6.64%-15.20%,and 25.73%-44.37% in 12 batch of D. huoshanense. These contents and ratio of peak areas of D-mannose to D-glucose (Am/Ag) were within the scope of D. officinale's; in addition, their average contents were basically the same with those in D. officinale (about 33%).Next,naringenin showed a good linear relationship within the range of 0.020 8-0.832 0 μg (r=0.999 9),and its average recovery was 101.96% (RSD 1.8%). The content of naringenin was 0.053 2-0.122 4 mg·g-1 (average value of 0.081 0 mg·g-1) in 11 batch of D. officinale, slightly higher than 0.040 3-0.090 0 mg ·g-1 (average value of 0.068 3 mg ·g-1) in 7 batch of D. huoshanense. All of these results of narinfenin did not reach the content lower limit in Ch.P. Conclusion: The method used to determinate the content of D-mannose and D-glucose is reproducible, and their sum content is possible to substitute the total polysccaride determination (with higher errors) in D. officinale; monosaccharide content determination can be used for quantitative quality control of D. huoshanense. However, it could not distinguish D. officinale and D. huoshanense by determining the contents of polysccharide,D-glucose,D-mannose and narinhenin, and shall be combined with other specificity methods for further identification.
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Objective: To study the antioxidation activities in vitro of a comment flavonoid component named vicenin Ⅱ(Apigenin 6,8-di-C-glucoside) in Dendrobii Officinalis Caulis from different origin places and investigate its effects on apoptosis of HepG2 cells. Method: The antioxidation activities in vitro of vicenin Ⅱ (0.005-1 g·L-1) were evaluated by 2,2-diphenyl-1-picrylhydrazyl (DPPH), salicylic acid and 2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid(ABTS) and copper ion reduction assays. Methye thiazolye telrazlium(MTT) assay was used to test the inhibitory effect of vicenin Ⅱ(12.5~100 μmol·L-1) on proliferation of 6 tumour cells in vitro. In subsequent apoptosis experiment, the concentration of vicenin Ⅱ was 75 μmol·L-1. The morphological changes of HepG2 cells were evaluated by Hoechst 33258 under fluorescence microscope; and the cell apoptosis rate was detected by flow cytometry with AnnexinV/PI apoptosis assay kit. The mRNA expressions of mitogen activated protein kinase (MAPK) pathway related apoptotic genes were detected by Real-time PCR assay. Result: The 1 g·L-1 vicenin Ⅱ showed 48.82% and 22.01% for DPPH scavenging rate and Cu2+ reduction rate respectively(P-1 vicenin Ⅱ showed 86.88% for ABTS scavenging rate(P-1 Vicenin Ⅱ, the cells survival rate was 45.69%(PPN-terminal kinase (JNK), and nuclear transcription factor (NF)-κB were increased(PConclusion: The general flavone glycosides component vicenin Ⅱ of Dendrobii Officinalis Caulis from different origins has a certain antioxidation effect and significant inhibitory effect on proliferation, and could induce apoptosis on HepG2 cells probably by regulating the expression of related genes in MAPK pathway and Bax/Bcl-2.