Cloning, expression analysis and enzyme activity verification of dihydroflavonol 4-reductase from Cistanche tubulosa (Schenk) Wight flower / 药学学报

Dihydroflavonol 4-reductase (DFR) plays an essential role in the biosynthesis of anthocyanin and regulation of plant flower color. Based on the transcriptome data of Cistanche tubulosa (Schenk) Wight, a full-length cDNA sequence of CtDFR gene was cloned by reverse transcription-polymerase chain reaction (RT-PCR). CtDFR contains an open reading frame (ORF) of 1 263 bp which encodes 420 amino acids with a predicted molecular weight of 47.5 kDa. The sequence analysis showed that CtDFR contains a nicotinamide adenine dinucleotide phosphate (NADPH) binding domain and a specific substrate binding domain. The expression analysis indicated that CtDFR was highly expressed in red and purple flowers, and the relative expression levels were 4.04 and 19.37 times higher than those of white flowers, respectively. The recombinant CtDFR protein was expressed in E.coli BL21 (DE3) using vector pET-28a-CtDFR and was purified. In vitro enzyme activity analysis, CtDFR could reduce three types of dihydroflavonols including dihydrokaempferol, dihydroquercetin, and dihydromyricetin to leucopelargonidin, leucocyanidin and leucodelphinidin. Subcellular localization analysis showed that CtDFR was mainly localized in the cytoplasm. These results demonstrate that CtDFR plays an important role in regulation of flower color in C. tubulosa and make a valuable contribution for the further investigation on the regulation mechanism of C. tubulosa (Schenk) Wight flower color.

Optimization of natural deep eutectic solvent extraction of phenylethanoid glycosides from Cistanche deserticola by response surface methodology coupled with genetic algorithm / 中国药房

OBJECTIVE To optimize the extraction technology fo r phenylethanol glycosides from Cistanche deserticola by natural deep eutectic solvents （NADESs），and to provide reference for the development and utilization of C. deserticola . METHODS The optimal NADESs was selected using total extraction efficiency of echinacoside ，acteoside and isoacteoside as indexes. Based on single factor test ，response surface methodology was used to select the optimal NADESs molar ratio ，the optimal NADESs water content ，the optimal liquid-solid ratio ；and the results were optimized by genetic algorithm . Using vitamin C （VC） as positive control ，the extraction effects of NADESs and traditional solvent （50％ methanol）were compared in respects of extraction efficiency and antioxidant activities. RESULTS The optimal extraction solution was NADES- 11 composed of 1， 4-butanediol and malonic acid. The optimal extraction technology was as follows as the molar ratio of 1，4-butanediol-malonic acid was 1 ∶ 2.5，water content of NADES- 11 was 18％，liquid-solid ratio was 30 mL/g，extraction time was 30 min and extraction temperature was 30 ℃. The extraction efficiency of NADES- 11 was significantly higher than that of 50％ methanol（P＜0.05）. IC 50 values of NADES- 11 extract（261.17 and 744.34 µg/mL）to 1，1-diphenyl-2-trinitrophenylhydrazine radical and hydroxyl radical were all lower than those of 50％ methanol extract （420.97 and 1 175.12 μg/mL）. Ascorbic acid equivalent antioxidant capacity of Δ 基金项目 内蒙古自治区科技创新引导项目（No.00120209）；内 NADES-11 extract（17.19 and 360.80 mg VC/g ）was higher 蒙古自治区自然科学基金资助项目 （No.2021MS08011）；内蒙古自治 than that of 50％ methanol extract （10.67 and 228.54 mg 区医疗卫生科技计划项目（No.202201367）；包头医学院“花蕾计划”项 VC/g）. CONCLUSIONS The optimized extraction process of 目（No.HL2021046） phenylethanol glycosides from C. deserticola using NADESs is ＊第一作者 硕士研究生。研究方向：中蒙药药效成分。E-mail： environmental，stable and feasible. dongjiani369@126.com

Antigen sparing with influenza virus vaccine formulated with crude polysaccharides from Cistanche deserticola Y. C.Ma adjuvant / 中华微生物学和免疫学杂志

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.

Immunomodulatory activity of ethanol extracts of cultivated Cistanche deserticola in Xinjiang / 国际生物医学工程杂志

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.

Rapid chemome profiling of Cistanche salsa using DI-MS/MS~(ALL) / 中国中药杂志

The current study aims to rapidly and comprehensively profile the chemical composition of Cistanche salsa using direct infusion coupled with MS/MS~(ALL)(DI-MS/MS~(ALL)). The C. salsa extract was directly imported into electrospray ionization(ESI) source of quadrupole time-of-flight(Q-TOF) mass spectrometer with an infusion pump at a flow rate of 10 μL·min~(-1). Acquisition program was applied under negative ionization polarity to collect one MS~1 spectrum(m/z 50-1 200), followed by 1 150 MS~2 spectra with precursor isolation window(m/z 1) amongst mass range m/z 50-1 200. After each MS~2 spectrum was matched to its precursor ion, putative identification was conducted through matching mass spectral data with literature and database. A total of 31 components were identified from C. salsa, including 9 phenylethanoid glycosides, 2 iridoids, 4 saccharides, 9 organic acids, and 7 other compounds, similar to those from C. tubulosa and C. deserticola. In conclusion, DI-MS/MS~(ALL), a facile and reliable analytical tool, can be employed for qualitative analysis of chemical constituents in C. salsa. The research offers a promising strategy to achieve rapid chemome profiling of herbal medicine and provides an alternative source of Cistanches Herba.

Analysis of the soil microbial community of Cistanche deserticola and Cynomorium songaricum in the saline-alkali soil of Ebinur Lake / 药学学报

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.

Protective effects of the total glycosides of Cistanche deserticola Y. C. Ma in alcoholic liver injury in mice / 药学学报

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.

Study on Separation and Immunocompetence of Water Extract of Polysaccharide from Cistanche tubulosa / 中国药房

OBJECTIVE：To isolate the water extract of polysaccharide from Cistanche tubulosa ，and to investigate their immunocompetence in vitro . METHODS ：AB-8 macroporous adsorption resin was used to decolorize C. tubulosa polysaccharide. The decolorization process was optimized by orthogonal test with retention rate and decolorization rate of polysaccharide as comprehensive score ，and using adsorption rate ，decolorization time ，sample concentration as factors. The verification tests were conducted. DEAE- 650M ion exchange column was used to separate the water extract of decolorized C. tubulosa polysaccharide. CCK-8 assay was used to detect the effect s of different concentration of polysaccharide （6.25-100 μg/mL）before and after isolation on the proliferation rate of mice macrophage RAW 264.7. Griess method and ELISA assay were adopted to detect the effects of low ， medium and high concentration of polysaccharide （12.5，25，50 μg/mL）on the release of NO ，IL-6 and TNF-α in LPS-induced RAW264.7 cells. RESULTS ：In the optimal decolorization process of AB- 8 macroporous adsorption resin ，the adsorption flow rate was 1.2 BV/h，the decolorization time was 9 h，and sample concentration was 25 mg/mL. The comprehensive scores of 3 times of verification tests were 63.43％，63.29％ and 63.34％，respectively，with an average of 63.35％（RSD＝0.11％，n＝3）. One neutral polysaccharide （CTZ）and 5 acid polysaccharides （CT1，CT2，CT3，CT4，CT5）were isolated from the polysaccharide of C. cistanche ，the contents were 299.2，168.0，123.2，121.6，54.4，11.2 mg/g. Compared with control group ，6.25-100 μg/mL CTZ （except for 6.25 μg/mL），CT2，CT4，CT5 and 6.25 μg/mL CTC（the polysaccharide before seperation ）could significantly increase the proliferation rate of RAW 264.7 cells（P＜0.05），while 6.25-100 μg/mL CT1，CT3 and 50 μg/mL CTC could decrease te proliferation rate of RAW 264.7 cells（P＜0.05）. Compared with LPS group ，the release of NO were decreased significantly in low，medium and high concentration groups of CTC ，CT2，CT3 and CT 5，CTZ low concentration group （P＜0.05），while were increased significantly in high concentration groups of CT 1 and CT 4 （P＜0.05）. The release of IL- 6 （except for CT 1 high concentration group and CT 5 low concentration group ）and TNF-α（except for CT 1 medium concentration group ）were decreased significantly in low ，medium and high concentration groups （P＜0.05）. CONCLUSIONS ：The optimized decolorization technology of macroporous adsorption resin is stable and feasible in the study. One neutral polysaccharide and 5 acidic polysaccharides can be isolated from water extract of C. tubulosa polysaccharides，among which CT 2 polysaccharide has stronger anti-inflammatory ability.

Study on Metabolic Transformation of Total Glycosides of Cistanche deserticola in Artificial Gastric and Intestinal Juice / 中国药房

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.

Effects of cistanche phenylethanol glycosides on the cardiac hypertrophy and PI3K/PKB signaling pathway in rats with pressure overload / 中国药理学通报

Aim To investigate the inhibitory effect of cistanche phenylethanol glycosides (CPhGs) on cardiac hypertrophy in rats caused by pressure overload and its related mechanism. Methods Male SD rats(n =70) were randomly divided into control group (Con), sham operation group (Sham), model group (Mod), positive control group (Vst), and different CPhGs dosage (125, 250, 500 mg • kg-1) groups. Cardiac ultrasound indexes, heart-weight to body-weight index (HWI), cardiac histopathological changes, and the area of myocardical cells (AMC) were detected. Plasma ET-1 and BNP levels were detected by Elisa, and protein expressions of phosphorylated PI3K(p-PI3K), PI3K, phosphorylated PKB (p-pKB) and PKB were detected by Western blot. Results Compared with Mod group, LVPWT, HWI, plasma ET-1, BNP and AMC decreased to different degrees. LVEDD, LVEF, LVFS, the protein expressions of myocardial tissues pPI3K and p-PKB increased to different degrees in CPhGs groups. Moreover, the indexes of CPhGs 250 and 500 mg • kg-1 groups were significantly improved compared to those of Mod group (P < 0. 05 or 0. 01). Compared with Vst group, there were no significant difference in CPhGs 500 mg • kg-1 group. Conclusions CPhGs could inhibit cardiac hypertrophy in rats induced by pressure overload, which might be related to the activation of PI3K/PKB signaling pathway.

Optimization of the Extraction Technology of Verbascoside from Cistanche tubulosa by Box-Behnken Response Surface Me- thodology / 中国药房

OBJECTIVE： To optimize the extraction technology of verbascoside from Cistanche tubulosa， and to provide reference for further development and comprehensive utilization of C. tubulosa. METHODS： The content of verbascoside in C. tubulosa was determined by HPLC. The determination was performed on  Inertsil-ODS-3V  column with  mobile phase consisted of methanol-0.2％ formic acid aqueous solution （40 ∶ 60， V/V） at  the flow rate  of  1 mL/min. The column temperature was 30 ℃， the detection wavelength was 330 nm， and the sample size  was 10 μL. Using extraction rate of verbascoside as index， soaking time， ethanol concentration， liquid-solid ratio， extraction time and extraction times were investigated by single factor tests. According to the results of above tests， ethanol concentration， liquid-solid ratio and extraction time were optimized by Box-Behnken response surface methodology. The verification tests were carried out on the optimized extraction technology. RESULTS： The linear range of verbascoside was 18.65-932.4 μg/mL. The optimal extraction technology included that ethanol concentration 63％， liquid-solid ratio 8 ∶ 1 （mL/g）， soaking for 2 h， extraction time 1.5 h， extracting for 2 times. The extraction rates of verbascoside in the three parallel verification tests were 78.21％， 76.95％， 79.34％， respectively. The relative errors of those to predicted value 76.76％ were 1.89％， 0.25％， 3.36％. CONCLUSIONS： The optimized extraction technology of verbascoside from C. tubulosa is stable and feasible， and is suitable for the extraction of verbascoside.

Network Pharmacology Exploration of the Mechanism of Cistanche deserticola in the Treatment of Osteoporosis / 中国药房

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.

Inhibitory effect of cistanches phenylethanol glycosides on lipopolysasccharide-induced acute lung injury in rats / 中国药理学与毒理学杂志

OBJECTIVE To study the effect of Cistanches phenylethanol glycosides (CPhGs) on acute lung injury in rats and the possible mechanism of action. METHODS Sixty SD rats were randomly divided into six groups: Normal control group, model group (lipopolysasccharide (LPS) 3 mg·kg-1], dexamethasone (Dex) positive control group (model+ Dex 2 mg·kg-1), model + CPhGs 125, 250 and 500 mg·kg-1 with 10 rats in each group. Each dose group of CPhGs was ig administered every day for 30 d, and the other three groups were ig administered with the same volume of distilled water for 30 d. The mode+Dex 2 mg·kg-1 group was ig given Dex 2 mg·kg-11 h before modeling. The rat model of acute lung injury was established after the rats in the other groups were intubated by the breath tube. Three hours after modeling, the rats were sacrificed by blood sampling from the abdominal aorta. The percentage of neutrophils in the whole blood, the activity of serum superoxide dismutase (SOD), glutathione (GSH) activity and the contents of malondialdehyde (MDA) and nitric oxide (NO) were detected. The contents of tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β) and IL-6 in alveolar lavage fluid were detected. The wet/dry mass ratio (W/D) of the middle lobe of the right lung was calculated and the histopathological changes of the posterior lobe of the right lung were observed by HE staining. RESULTS Compared with normal control group, the percentage of neutrophils in the whole blood, W/D in the middle lobe of the right lung, the contents of MDA and NO in the serum were increased (P<0.05), but SOD, GSH, TNF-α and IL-6 were decreased in the model group (P<0.05). Compared with the model group, the percentage of neutrophils in the whole blood and W/D in right middle lobe of the right lung in the group of model+CPhGs 500 mg·kg-1 was decreased (P<0.05), but the contents of TNF-α and IL-6 in alveolar lavage fluid of rats decreased significantly, and histopathological observation showed that inflammation was alleviated to varying degrees. The activities of SOD and GSH in rat serum of the model + CPhGs 500 mg·kg-1 group were increased significantly (P<0.05), while the contents of MDA and NO were decreased significantly (P<0.05). CONCLUSION CPhGs have a protective effect against acute lung injury induced by LPS in rats, and its mechanism may be related to its antioxidant effect, alleviation of pulmonary edema and reduction of the release of inflammatory factors.

Effect of drought stress on accumulation of two respective phenylethanoid glycosides in tissue culture of Cistanche tubulosa / 中草药

Objective: To establish the plant tissue culture system of Cistanche tubulosa, and determine the effect of drought stress on accumulation of two respective phenylethanoid glycosides in it. Methods The major chemical constituents of C. tubulosa by tissue culture were analyzed by HPLC-UV and HR-MS. The cell growth curves were also determined. In addition, the effects of drought stress on the phenylethanoid glycosides (echinacoside and acteoside) content in the tissue culture system of C. tubulosa were also studied by using NaCl, mannitol and PEG6000 as osmotic regulators, respectively. Results:Chemical constituents analyses revealed that callus and suspension cultures of C. tubulosa could produce the respective phenylethanoid glycosides of echinacoside and acteoside as in wild plant; Cell growth curves indicated that 30 d were the optimum culture period of callus culture; The cell growth rate and the accumulation of echinacoside and acteoside were mostly inhibited when the callus cells were under drought stress induced by NaCl or mannitol. Meanwhile, the accumulation of echinacoside and acteoside in cell suspension culture of C. tubulosa could be effectively enhanced by treatment with PEG6000. The maximum biomass of echinacoside and acteoside could reach to (1.07 ± 0.10) g/L and (0.12 ± 0.01) g/L 15 d after induction, respectively. And their contents were 20.94% and 2.27% separately based on the cell dry weight (DW) after 15 d of treatment with 6% PEG6000, which were 1.29 and 1.19 fold higher than the control group. Conclusion:Drought stress induced by PEG6000 could effectively enhance the accumulation of echinacoside and acteoside in cell suspension culture of C. tubulosa.

Extraction parameters and drying methods of Cistanche deserticola / 中草药

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.

Parasitic relationship of Cistanche deserticola and host-plant Haloxylon ammodendron based on genetic variation of host / 中草药·英文版

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.

Process optimization for extraction and purification of polysaccharides from Cistanche deserticola / 中国中药杂志

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.

Neuroprotective effects and mechanism of ethanol extract of Cistanche tubulosa against oxygen-glucose deprivation/reperfusion / 中国中药杂志

To investigate the inhibitory effects and mechanism of Cistanche tubulosa ethanol extract( CTEE) against oxygen-glucose deprivation/reperfusion( OGD/R)-induced PC12 cells neuronal injury. In this study,OGD/R-induced PC12 cells were used to explore the neuroprotective effects of CTEE( 12. 5,25,50 mg·L-1) by detecting cell viability with MTT assay,apoptosis with AO/EB and Hoechst 33258,mitochondrial membrane potential changes with JC-1 staining,mitochondrial oxidative stress with MitoSOX staining,as well as the apoptosis-related protein expression( PARP,cleaved PARP,caspase-3,cleaved caspase-3,Bax,Bcl-2) with Western blot. RESULTS:: showed that CTEE effectively protected OGD/R-induced neuronal injury and increased the survival rate of PC12 cells.AO/EB and Hoechst 33258 staining showed that CTEE could effectively inhibit apoptosis. Moreover,JC-1 and MitoSOX staining results showed that CTEE decreased mitochondrial stress and mitochondrial membrane potential imbalance in PC12 cells in a concentration-dependent manner. Meanwhile,CTEE could obviously suppress the activation of key proteins in mitochondrial apoptosis pathway such as caspase-3 and PARP,and significantly inhibit the rise of Bax and down-regulation of Bcl-2. In conclusion,CTEE has obvious protective effects on OGD/R-induced PC12 cells neuronal injury,potentially via inhibiting mitochondrial oxidative stress and apoptosis-related signaling pathway.

Comparative studies of three Cistanche speices based on UPLC specific chromatogram and determination of main components / 中国中药杂志

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.

Therapeutic effect of cistanche deserticola ethanol extraction in rats with osteoporosis induced by ovariectomy and its mechanism / 吉林大学学报(医学版)

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.