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Objective:To investigate the antigen-sparing effects of crude polysaccharides from Cistanche deserticola Y. C.Ma (CPCD) for influenza virus vaccine (IVV). Methods:ICR mice were immunized subcutaneously with CPCD combined with different doses of IVV (0.01 μg and 0.1 μg). Hemagglutinin inhibition (HI) assay was used to detect HI titers in serum samples. Indirect ELISA was performed to detect the levels of specific IgG antibodies and their subtypes in serum samples. The proliferation of splenic lymphocytes was detected by MTT assay. The percentages of CD4 + , CD8 + and CD44 + T cells and the levels of IFN-γ in splenic cells isolated from the vaccinated mice were analyzed by flow cytometry. Results:CPCD significantly increased HI titers (234.67±47.70 vs 149.33±47.70, P<0.05), promoted the production of IgG ( A450 value: 1.16±0.63 vs 0.30±0.21, P<0.05) and IgG1 ( A450 value: 1.09±0.60 vs 0.26±0.21, P<0.05) and enhanced splenic lymphocyte proliferation ( P<0.05). CPCD also significantly up-regulated the expression of CD4 + [(41.97±4.58)% vs (25.43±1.48)%, P<0.05], CD8 + [(12.67±0.33)% vs (9.02±1.07)%, P<0.05], CD4 + CD44 + [(11.77±0.69)% vs (8.64±0.71)%, P<0.05] and CD8 + CD44 + [(6.70±0.67)% vs (4.66±0.39)%, P<0.05] T cell subsets as well as the secretion of IFN-γ in CD4 + [(1.36±0.07)% vs (0.87±0.06)%, P<0.05] and CD8 + [(2.09±0.20)% vs (1.42±0.08)%, P<0.05] T cells. In addition, there was no significant difference between CPCD combined with low-dose IVV group and high-dose IVV alone group ( P>0.05), implying a 10-fold antigen sparing. Conclusions:CPCD, as an adjuvant for influenza virus vaccine, could enhance humoral and cellular immune responses and reduce antigen dose, which might be a potential adjuvant for seasonal or pandemic influenza vaccines.
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Objective:To explore the immunomodulatory activity of ethanol extract of cultivated Cistanche deserticola (EECCD) in Xinjiang. Methods:Ovalbumin (OVA) was used antigen, ICR mice were divided into 9 g/L NaCl group (blank control group), EECCD group (1 200 μg EECCD), OVA group (10 μg OVA), low-dose EECCD/OVA group (400 μg EECCD+10 μg OVA), medium-dose EECCD/OVA group (800 μg EECCD+10 μg OVA), high-dose EECCD/OVA group (1 200 μg EECCD+10 μg OVA) and aluminum adjuvant (Alum)/OVA group (200 μg Alum+10 μg OVA). Mice were immunized subcutaneously, and the immunization was strengthened once 14 days after the initial immunization. The level of splenocyte proliferation was determined by thiazolyl blue tetrazolium bromide (MTT) method, and interferon γ (IFN-γ) and interleukin-4 (IL-4) in CD4 + T cell, dendritic cells (DCs) surface markers and CD4 +CD25 +Foxp3 + Treg were evaluated by fluorescence-activated cell sorting (FACS). Results:Three dose of EECCD can enhance OVA-specific IgG titers in serum. The antibody titer in medium-dose EECCD/OVA group was 250 000, which was the same as that in the Alum/OVA group. The medium-dose EECCD/OVA significantly improve IgG1 and IgG2a (both P<0.01). Therefore, the medium dose EECCD was selected as the best dose. MTT results displayed that splenocyte proliferation were significantly stimulated by medium-dose EECCD/OVA ( P<0.05), and the levels of IL-4 and IFN-γ in CD4 + T cells were promoted in groups administered with medium-dose EECCD/OVA (both P<0.01). Furthermore, medium-dose EECCD/OVA significantly up-regulated the levels of CD40, CD80, CD86 and major histocompatibility complex class Ⅱ (MHCⅡ) on DCs and down-regulated the frequency of CD4 +CD25 +Foxp3 + Treg (all P<0.05). Conclusions:EECCD has good immunomodulatory activity, can promote Th1-biased response, and has the therapeutic potential for the prevention of diseases.
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To explore the characteristics of soil microbial communities of Cistanche deserticola and Cynomorium songaricum, two typical parasitic medicinal plants that live in an extreme saline alkali environment, 16S PCR was used to sequence the soil microbial communities of C. deserticola and C. songaricum in Ebinur Lake, Xinjiang. Redundancy analysis and correlation analysis were carried out based on the abundance of core microbiome and ecoclimatic factors. The results show that the diversity of the soil microbial community of C. deserticola was significantly higher than that of C. songaricum. The core microbial groups of C. deserticola and C. songaricum were Marinomona, Halomonadaceae, Rhizobiales, Halomonas, and Acidimicrobiales. Six specific biomarkers were identified as Micrococcacea, Echinicola, Glutamicibacter, Galbibacter, Pseudoalteromonas, and Marinobacterium_ rhizophilum. The results of redundancy analysis and correlation analysis show that the average temperature in the driest season and the average temperature in the coldest season, and the clay content and soil texture classification were the main ecological factors affecting the composition of these soil microbial communities. This study provides a theoretical basis for finding molecular markers of C. deserticola and C. songaricum and promoting the quality of C. deserticola and C. songaricum.
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Modern pharmacological studies have shown that Cistanche deserticola (C. deserticola) has a protective effect on the liver, but its active fraction and mechanism are not clear. In order to identify the effective fraction of C. deserticola Y. C. Ma, an acute alcoholic liver injury model in mice was established with 56-proof Erguotou and different fractional extracts of C. deserticola Y. C. Ma (total glycosides, polysaccharides, and oligosaccharides) were administered. After 14 days of oral administration, liver pathology and lipid deposition were measured and the expression of nuclear factor E2-related factor (Nrf-2), kelch-like ECH-associated protein-1 (Keap-1), and plasmalemma vesicle-associated protein-1 (PV1) were measured by immunofluorescence. The levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), endotoxin (ET), diamine oxidase (DAO), and D-lactic acid (D-LA) in serum, and superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT), and malondialdehyde (MDA) in liver were measured by ELISA. All animal experiments were carried out with approval of the Experimental Animal Welfare Ethics Committee of the Peking University Health Science Center. The results show that the total glycosides of C. deserticola Y. C. Ma (400 mg·kg-1) could decrease liver pathology, decrease serum endotoxin, diamine oxidase, and D-lactic acid, and reduce hepatic lipid deposition. Total glycosides also promoted Nrf-2 transfer into the nucleus and decreased the expression of Keap-1 and PV1. In summary, the total glycosides of C. deserticola Y. C. Ma had a protective effect in acute alcoholic liver injury and the mechanism may be related to the activation of the Nrf-2/Keap-1 pathway, improvement of intestinal wall integrity, and inhibition of the transport of harmful substances into the liver.
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ABSTRACT OBJECTIVE：To study the metabolic transformation of total glycosides of Cistanche deserticola in artificial gastric and intestinal juice，and to speculate its metabolic transformation pathway in vivo. METHODS：UPLC/Q-TOF-MS was adopted. The determination was performed on ACQUITY UPLC BEH column with mobile phase consisted of 0.2％ formic acid water-acetonitrile（gradient elution）at the flow rate of 0.2 mL/min. The detection wavelength was set at 330 nm，and column temperature was 25 ℃. The ion source was electrospray ion source，and mass to charge ratio（m/z）was 50→1 000. In the positive and negative ion mode，the metabolic components of the total glycosides of C. deserticola in artificial gastric and intestinal juice were identified analysis，and combined with the literature，the metabolic pathway of total glycosides of C. deserticola in artificial gastric and intestinal juice was speculated. RESULTS：After the total glycosides of C. deserticola were metabolized by artificial gastric juice，and a total of 69 components were estimated，including 14 prototype components （such as Mustard aldehyde glucoside，daucosstorol） and 55 metabolic components （such as Methyl-O-Kankanoside J，Methyl-O-Kankanoside E），it is speculated that its metabolic pathways were methylation，demethylation，hydroxylation，methoxylation，acetylation，sulfation，and glucuronidation. After the total glycosides of C. deserticola were metabolized by artificial intestinal juice，a total of 90 components were estimated，including 4 prototype components（such as Kankanoside M，Kankanoside L）and 86 metabolic components（such as Methyl-O-Kankanoside， Methyl-O-Kankanoside E）. It was speculated that its metabolic pathways were methylation， demethylation，hydroxylation，dehydroxylation，methoxylation，acetylation，sulfation and glucuronidation. CONCLUSIONS：This study preliminarily speculates that the total glycosides of C. deserticola may be metabolized by methylation，demethylation， hydroxylation and other metabolic pathway in artificial gastrointestinal juice，which may provide reference for the in vivo metabolic transformation of total glycosides of C. deserticola.
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In this study, taking Cistanche deserticola in Xinjiang as the experimental material, the optimal process for extracting polysaccharides from C. deserticola with water extraction was studied by using single factor and orthogonal experiment. Its effects on protein removal and polysaccharides retaining were investigated by using Sevag, enzymatic method or combination of these two methods, so as to determine the optimal method for protein removal from polysaccharides of C. deserticola; the decolorization and purification methods such as macroporous resin of AB-8 and activated Carbon were used to determine the optimal process. The results showed that the extraction rate of polysaccharides from C. deserticola was 18.40% during the optimal process of the water extraction as follows: extraction temperature 75 ℃, extraction time 165 min and solid-liquid ratio 1∶55. The protein removal rate can reach 31.40% and polysaccharide retention rate can reach 96.00% under the optimal protein removal process: temperature 50 ℃, time 2 h, and papain dosage 0.2%. The decolorization rate of activated Carbon and macroporous resin called AB-8 was 80.37% and 86.43%, and the recovery rate of polysaccharides was 77.05% and 91.93%, respectively, suggesting that macroporous resin was more suitable for decoloration. Macroporous resin named AB-8 increased the purity of the polysaccharide crude extract from 67.70% to 84.80% under the following conditions: concentration of the sample 4 g·L~(-1), concentration of the eluent 60% ethanol, and the flow rate 1 mL·min~(-1), showing significant purification effect.
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Cistanche , Chemistry , Plant Extracts , Chemistry , Polysaccharides , Temperature , WaterABSTRACT
Objective: The optimum extraction process parameters of Cistanche deserticola were selected to study the effects of different drying methods on five phenylethanoid glycosides. Methods: Single factor screening combined with Box-Behnken response surface method was used to optimize the extraction process. After optimal conditions were extracted, HPLC method was used to detect the content of echinacoside, cistanche A, verbascoside, isoacteoside, and 2’-acetylacteoside in different drying methods, and one-way ANOVA, cluster analysis, principal component analysis and close value analysis were used to analyze the content of five phenylethanoid glycosides to choose the best drying method. Results: Optimal extraction process was as following: methanol volume fraction was 55.14%, liquid to material ratio was 46.39, extraction time was 38.50 min. Cluster analysis, principal component analysis, and close value analysis showed that the quality of C. deserticola obtained by freeze-drying method was the best, followed by drying at 80 ℃ and the lowest at 40 ℃. Conclusion: Using this process to extract C. deserticola, the five phenylethanoid glycosides are completely and fully extracted. Although the freeze-drying method of C. deserticola has the highest active ingredient retention, from the production point of view, the 80 ℃ drying method can achieve a balance of cost and efficiency.
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Objective: Cistanche deserticola is a famous and endangered medicinal plant that is parasitic upon Haloxylon ammodendron with rather low parasitic rates. It is important to find high affinity germplasms for increasing the survival of C. deserticola. However, little is known in genetic variation and high affinity populations of H. ammodendron in China. Methods: In this study, 98 accessions of H. ammodendron seeds were collected from five regions covering almost the entire natural distribution of H. ammodendron in China. Their genetic variations were analyzed using AFLP and ITS by the maximum parsimony method, and a dendrogram was constructed using the unweighted pair-group method with arithmetic average (UPGMA). The parasitic rates of C. deserticola on different accessions of H. ammodendron were calculated in the field experiment. Results: Both AFLP and ITS methods consistently revealed that there was a high level of genetic diversity in the natural populations of H. ammodendron. Hierarchical population structure analysis uncovered a clear pattern that all populations were grouped into three main clusters, and eight populations from eastern region were genetically clustered together. These regions were significantly differentiated (P < 0.05), 13.10% of variation occurred among populations, and 86.90% within populations was revealed by analysis of molecular variance (AMOVA). The populations of Inner Mongolia had the highest parasitic rates followed by Ganjiahu Reserve and Yongning Plantation for the top three, which were not completely related to the genetic variation. Conclusion: Genetic characteristics of H. ammodendron in China were clarified and the order of affinity of different populations was given, which were primers for discovering high affinity germplasms.
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OBJECTIVE： To study the mechanism of Cistanche deserticola in the treatment of osteoporosis by network pharmacology. METHODS： The active components of C. deserticola were retrieved and obtained by TCM system platform （TCMSP）. Reverse molecular docking server DRAR-CPI and related databases GeneCards and OMIM were used to screen the target of C. deserticola active ingredients in the treatment of osteoporosis. The “component-target”network of C. deserticola was constructed by Cytoscape software， and the interaction between targets was plotted by String database and Cytoscape software. The combination activity of target and active ingredient was evaluated via molecular docking with Systems Dock WebSite server. GO classification and enrichment analysis and KEGG pathway enrichment analysis were conducted for target genes using DAVID database. RESULTS： Totally 13 active ingredients were screened out from C. deserticola， such as verbascoside， leonurine， geniposidic acid. There were 43 active ingredient-treated potential targets， such as RUNX2， VEGF， IL-6， BGP， TNF. Multiple signaling pathways were involved in target action， such as WNT （Wingless/Integrated）， VEGF， TNF. CONCLUSIONS： This study preliminarily explores and validates the main targets and pathways of C. deserticola in the treatment of osteoporosis， which lay the foundation for further study of its mechanism.
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Based on UPLC specific chromatogram and determination of seven main components,this study aimed at evaluating the quality of Cistanche deserticola,C. tubulosa and C. sinensis. Echinacoside,cistanoside A,verbascoside,tubuloside A,isoacteoside,2'-acetylacteoside,tubuloside B were used as reference substances. UPLC analysis was performed on a Waters ACQUITY UPLC HSS T3 column( 2. 1 mm×100 mm,1. 8 μm). The mobile phase was acetonitrile-0. 08% trifluoroacetic acid solution. The flow rate was0. 3 mL·min-1,and the injection amount was 10 μL. The column temperature was 40 ℃,and the detection wavelength was 330 nm.The UPLC specific chromatograms were processed with ChemPattern software. UPLC specific chromatograms of C. deserticola and C.tubulosa from different samples were of high similarity,but the similarities of their counterfeit C. sinensis were less than 0. 06. Both of cluster and principal component analysis can distinguish certified products and counterfeits. The content ratios of echinacoside/verbascoside and verbascoside/isoacteoside were quite different between C. deserticola and C. tubulosa,which had distinct significance.The UPLC specific chromatogram and contents of seven main components can provide a basis for quality evaluation of Cistanches Herba.
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Chromatography, High Pressure Liquid , Cistanche , Chemistry , Classification , Drugs, Chinese Herbal , Reference Standards , Phytochemicals , Principal Component AnalysisABSTRACT
Objective To study the effects of aqueous extracts of cultivated Cistanche deserticola Y. C. Ma (AECCD) on T cell responses and the duration of antibody response and to investigate its immunoen-hancing activities in mice. Methods Two batches of female ICR mice were used in this study with 30 from each batch. Each batch of mice was randomly divided into six groups (n=5). Low, medium and high doses of AECCD in combination with ovalbumin ( OVA) were used to set up three experimental groups, while 0. 9% NaCl, OVA alone and aluminium adjuvant were respectively used as blank, negative and positive controls. All mice were intramuscularly injected twice at an interval of two weeks. Flow cytometry was used to detect the ex-pression of T lymphocyte subsets, cytokines and surface molecules of dendritic cells (DC). Indirect ELISA was used to detect IgG antibody levels. Results AECCD could significantly increase the percentage of CD4+and CD8+T lymphocytes in spleen (P<0. 05), up-regulate the expression of CD4+CD44+and CD8+CD44+effector T lymphocytes (P<0. 05), promote the secretion of IFN-γ in T lymphocytes and enhance the expression of CD40 and CD80 on the surface of DC (P<0. 05). ELISA results showed that high-dose AECCD could significantly prolong the duration of IgG antibody response induced by OVA (P<0. 05). Conclusion AECCD could en-hance the T lymphocyte immune response induced by OVA and keep it maintained at a high level, which might help to improve the body′s immune response.
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Objective To compare the immunopotentiating effects of polysaccharides extracted from wild/cultivate Cistanehe deserticola (WCDPS/CCDPS) in Xinjiang. Methods ICR mice were subcu-taneously injected twice with different doses(low,medium and high) of WCDPS and CCDPS in combination with ovalbumin (OVA). OVA-specific antibody IgG,as well as IgG1 and IgG2a subtypes, was determined by ELISA. OVA-specific lymphocyte proliferation was measured by MTT. Expression of CD4+T and CD8+T cells was analyzed by flow cytometry. Results Both WCDPS and CCDPS could significantly improve the production of OVA-specific IgG,IgG1 and IgG2a,promote the proliferation of OVA-specific lymphocytes and increase the expression of CD4+T and CD8+T cells(all P<0.05) with no significant difference between them at the same dosages (P>0.05). WCDPS and CCDPS had no influence on the body weight of mice after im-munization. Conclusion WCDSP and CCDPS could significantly enhance the OVA-specific humoral and cellular immune responses with no statistical difference and are characterized by high safety.
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Objective: To explore the therapeutic effect of cistanche deserticola ethanol extraction (CDE) in the rats with osteoporosis induced by ovariectomy, and to clarify its mechanism. Methods: A total of 72 female SD rats were randomly divided into control, model, estradiol (0. 3 g · kg-1), low, middle and high doses (0. 5, 1.0 and 2. 0 mg · kg-1) of CDE groups, and 12 rats in each group. The rat ovaries were removed by operation in the other groups to establish the osteoporosis models, while the same size of fat of the rats in control group was excised. 5 d later, the rats in estradiol group were hypodermicly injected with estradiol, and the rats in CDE groups were orally administrated with corresponding doses of CDE. The same volume of diluted water was given to the rats in control and model groups. After 20 weeks, the density of right femur of the rat was detected with the method of ratio of weight and size; the mechanics index of left femur of the rat was analyzed by bone granulometer; the levels of serum alkaline phosphatase (ALP) and osteocalcin were measured by ELISA; the levels of serum Ca2 and P were detected by automatic biochemical analyzer; the morphology of uterus tissue was observed by HE staining. Results: Compared with model group, the densities of right thighbone of the rats in estradiol group and low, middle and high doses of CDE groups were significantly increased (P0. 05). Compared with model group and low dose of CDE group, the maximum bending forces, the maximun strains, and the bone rigid coefficients in middle and high doses of CDE groups were remarkably increased (P0. 05). Compared with model group, the morphology of uterus tissue of the rats in low, middle and high doses of CDE groups was improved at different degrees. Conclusion: CDE has therapeutic effect on osteoporosis through increasing the levels of serum ALP, osteocalcin and Ca' of the rats.
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Objective:To explore the therapeutic effect of cistanche deserticola ethanol extraction (CDE) in the rats with osteoporosis induced by ovariectomy,and to clarify its mechanism.Methods:A total of 72 female SD rats were randomly divided into control,model,estradiol (0.3 g · kg-1),low,middle and high doses (0.5,1.0 and 2.0 mg · kg-1) of CDE groups,and 12 rats in each group.The rat ovaries were removed by operation in the other groups to establish the osteoporosis models,while the same size of fat of the rats in control group was excised.5 d later,the rats in estradiol group were hypodermicly injected with estradiol,and the rats in CDE groups were orally administrated with corresponding doses of CDE.The same volume of diluted water was given to the rats in control and model groups.After 20 weeks,the density of right femur of the rat was detected with the method of ratio of weight and size;the mechanics index of left femur of the rat was analyzed by bone granulometer;the levels of serum alkaline phosphatase (ALP) and osteocalcin were measured by ELISA;the levels of serum Ca2+ and P were detected by automatic biochemical analyzer;the morphology of uterus tissue was observed by HE staining.Results:Compared with model group,the densities of right thighbone of the rats in estradiol group and low,middle and high doses of CDE groups were significantly increased (P＜0.05 or P＜0.01);there were no differences between low,middle and high doses of CDE groups (P＞0.05).Compared with model group and low dose of CDE group,the maximum bending forces,the maximun strains,and the bone rigid coefficients in middle and high doses of CDE groups were remarkably increased (P＜ 0.05),while the levels of ALP were decreased (P＜ 0.05).Compared with model group,and low and middle doses of CDE groups,the level of osteocalcin in high dose of CDE group was significantly decreased (P＜0.05).Compared with model group,the levels of Ca2+ in serum of the rats in low,middle and high doses of CDE groups were significantly increased (P＜0.05);there were no significant differences between low,middle and high doses of CDE groups (P＞0.05).Compared with model group,the morphology of uterus tissue of the rats in low,middle and high doses of CDE groups was improved at different degrees.Conclusion:CDE has therapeutic effect on osteoporosis through increasing the levels of serum ALP,osteocalcin and Ca2+ of the rats.
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In order to clarify the chemical constituents of Cistanche deserticola cultured in Tarim desert, a systematically phytochemical investigation was carried out. The chemical constituents were isolated by column chromatography, such as silica gel, Sephadex LH-20, MCI gel, ODS and semi-preparative HPLC, and their structures were determined on the basis of MS, NMR spectroscopic analysis, and comparison with literature data. Four compounds were isolated from the 85% ethanol extract of the stems of C. cultured in Tarim desert. Their structures were identified as cis-tubuloside (1), cis-cistanoside (2), cis-cistanoside J (3), and cis-isocistanoside C(4). Compounds 1-4 were four new cis-phenylethanoid glycosides. Herein, we firstly report the ¹H, ¹³C-NMR data of the new compounds(1-4) for the first time. This study will provide the scientific evidence for comprehensively analyzing the chemical constituents of C. deserticola cultured in Tarim desert.
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As a holoparasitic plant, Cistanche deserticola is one of the two original sources of Cistanches Herba that is one of the most famous tonic medicines, in Chinese Pharmacopoeia. The succulent stems are used for medicinal usage, whereas those lignified stems as well as flowers of less pharmacological importance are usually deserted, suggesting extensive resource waste. Herein, chemical characterization of the flowers along with lignified stems was conducted using HPLC-IT-TOF-MS aiming to explore the medicinal valu of those non-medicinal parts. Following ultrasonication-assisted extraction with 50% aqueous methanol, either flower or lignified stem extract was subjected onto LC-IT-TOF-MS equipped with a Capcell core ADME column to acquire both MS¹ and MSº spectra, and gradient elution was carried out with combinatory 0.1% aqueous formic acid and acetonitrile. Both positive and negative ionization polarities were deployed, resulting in the observation of 62 components, in total. Thirty-nine signals were structurally annotated, including phenylethanoid glycosides, iridoids, lignans and saccharides according to matching with authentic components and literature information, as well as applying the proposed mass fragmentation rules. A total of 62 ones were putatively identified. Above all, lignified stems and flowers should not the qualified substitutes for the succulent stems attributing to the significant differences between the medicinal portion and those parts with less medicinal values.
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(CD) is one of the two authoritative source plants of Cistanches Herba, a well-known medicinal plant. Herein,H NMR spectroscopy was employed to characterize the chemical profile and to distinguish the different parts, as well as to propose a new processing workflow for CD. Signal assignment was achieved by multiple one and two dimensional NMR spectroscopic techniques in combination with available databases and authentic compounds. The upper parts of the plant were distinguished from the lower parts by combiningH NMR spectroscopic dataset with multivariate statistical analysis. A new processing method that hyphenated steaming with freeze-drying, was demonstrated to be superior to either steaming coupled with oven-drying or direct freeze-dryingholisticH NMR-based metabolomic characterization. Phenylethanoid glycosides, mainly echinacoside and acteoside, were screened out and confirmed as the chemical markers responsible for exhibiting the superiority of the new processing workflow, whereas serial primary metabolites, especially carbohydrates and tricarboxylic acid cycle metabolites, were found as the primary molecules governing the discrimination between the upper and lower parts of the plant. Collectively,H NMR spectroscopy was demonstrated as a versatile analytical tool to characterize the chemical profile and to guide the in-depth exploitation of CD by providing comprehensive qualitative and quantitative information.
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This study aims to investigate the targets and targets-involved mechanism for the macrophage activation of low molecular weight saccharides from Cistanche deserticola (LMSC). The phagocytic activity and NO release of RAW264.7 cells were detected, and results showed that LMSC exerts immune activation effect by significantly increasing the phagocytic activity and NO release. LMSC-conjugated epoxy-activated sepharose beads were prepared as affinity reagent to capture the target proteins. Twenty-four proteins such as Eef2 were identified by LC-MS/MS analysis. Pathway enrichment analysis showed LMSC activated RAW264.7 cells by regulating Fcgamma receptor dependent phagocytosis, TNF-alpha NF-κB signaling pathway, glycolysis/gluconeogenesis and the citric acid cycle and respiratory electron transport pathway.
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To investigate the immune activation effect and mechanism of low molecular weight saccharides from Cistanche deserticola(LMSC) on mouse peritoneal macrophages, RAW264.7 cells. The RAW264.7 cells were divided into the normal control group, LPS positive control group, and LMSC treatment groups. The RAW264.7 cells were treated with various concentrations of LMSC from 3.91 to 62.5 g•L ⁻¹. The neutral red assay was employed to detect the phagocytic activity of macrophages. NO release was detected by using NO kit, and macrophage activation associated protein expression levels (TNF-α, IL-6, IKKβ, p-IKKβ, IκBα, p-IκBα, NF-κB, and p-NF-κB) were detected by Western blot. Results showed that LMSC had an activation effect on macrophages; it can significantly increase the release of NO in RAW264.7 cells and promote the expression of cytokines TNF-α and IL-6. Moreover, LMSC significantly increased the phosphorylation of IKKβ, IκBα, and NF-κB p65. Furthermore, mannitol's one of the main constituents in LMSC significantly enhanced the phagocytic activity of macrophages. These results showed that LMSC could activate macrophages by up-regulating the NF-κB signaling pathway, and mannitol may be one of the main active components in LMSC.
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Objective To investigate the contents of major five functional components (echinacoside, verbascoside, galactitol, betaine, soluble polysaccharide, and extractums) in Cistanche deserticola harvested in spring and autumn from genuine producing area in Alax Banner of Inner Mongolia. Methods HPLC-UV was applied to measuring the contents of echinacoside and verbascoside. HPLC-ELSD was used for determining the contents of galactitol and betaine. UV-VIS was utilized for analyzing the content of soluble polysaccharide. Results The index components of two samples' harvested in spring and autumn were all up to the standard of Chinese Pharmacopoeia. The samples harvested in spring contain 12.21 mg/g, which was twice of the autumn samples and fourfold of the standard of Chinese Pharmacopoeia. Based on verbascoside and betaine, the content of spring samples was significantly higher than the autumn samples, which was up to thirtyfold and it had great fluctuation among samples; Based on soluble polysaccharide, the content of spring and autumn samples were all at a high level, especially autumn samples was up to 13.7%; Based on galactitol and extractums, the content of autumn samples was significantly higher than the spring samples. Conclusion The content of verbascoside in C. deserticola has great fluctuation among samples. C. deserticola, that is rich in galactitol and soluble polysaccharide that authentic inner quality characteristics are "Glossy, Heavy, Fleshy, Quality soft, Sweet". Nevertheless, the ample galactitol and soluble polysaccharide is the material basis of quality formation, it is more reasonable to add the soluble polysaccharide as one of the index component to evaluate its quality characteristics. The standard of Chinese Pharmacopoeia points out the moisture limit of succulent herb is 10% may not be reasonable. The further improvement of the standard of C. deserticola's quality or separation of C. deserticola was discussed in this manuscript.