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.

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.

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.

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.

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.

Chemome comparison between cultivated and wild Cistanche tubulosa using ¹H-NMR spectroscopy / 中国中药杂志

As a famous tonic medicine, Cistanche tubulosa has been honored as "ginseng of the deserts" for centuries. Aiming to address the resource shortage as well as the wild resource protection towards this herbal medicine, wide cultivation has been achieved in the southern Xinjiang. Herein, in-depth chemome comparison was conducted between cultivated and wild plants using ¹H-NMR spectroscopy that is capable of comprehensively providing qualitative and quantitative information of given complicated matrices. Multivariate statistical analysis was employed to process the dataset as well as to consolidate that the cultivated plants are comparable to those wild ones in term of chemome. ¹H-NMR spectra of both wild and cultivated plants were acquired in parallel after extraction. Following direct overlaying, great similarity occurred between these two groups. A total of 28 compounds were tentatively identified by referring to authentic compounds together with those available databases, such as HMDB and BMRB. Following principal component analysis, none significant difference was observed between wild and cultivated groups. Above all, from the viewpoint of chemical profile, the cultivated plants were almost equal to the wild plants; therefore, the cultivated plants are able to take the load of wild plants in clinical usage. Moreover, ¹H-NMR spectroscopy is a promising tool for chemical profiling traditional Chinese medicines because of the potential towards simultaneously exhibiting both quantitative and qualitative information for complicated matrices.

Effects of phenylethanol glycosides from Cistanche tubulosa on proliferation of rat HSC induced by PDGF-BB and its mechanism / 中国药理学通报

Aim To investigate the effect of phenyle-thanol glycosides from Cistanche tubulosa(CPhGs) on the proliferation and activation of rrPDGF-BB induced HSC and their target points for resisting hepatic fibro-sis,to elucidate the molecular mechanism in molecular level, and provide basic data for the further develop-ment of new drugs. Methods HSCs were cultivated by CPhGs with different concentrations ( 0 , 3. 91 , 7. 81 , 15. 63 , 31. 25 , 62. 50 , 125. 00 , 250. 00 , and 500 mg ·L-1 ) and IC50 of CPhGs was determined. CPhGs with different concentrations ( 25 , 50 , 75 , 100 mg · L-1 ) were selected, and after the cells were stimulated with rrPDGF-BB, cell proliferation was determined by MTT. ERK1/2 ,α-SMA, c-fos, c-jun and Collagen I mRNA and Erk1/2 ,P-Erk1/2 and CollagenⅠprotein ex-pressions were assayed by RT-PCR and Western blot. Results CPhGs of ( 50 ~100 ) mg · L-1 concentra-tions groups could effectively inhibit rrPDGF-BB-medi-ated proliferation(P0. 05 ) . CPhGs of ( 25 ~100 ) mg · L-1 concentrations groups could inhibit ERK1/2 ,α-SMA,c-fos, c-jun and CollagenⅠmRNA levels, and also ob-viously inhibited Erk1/2 ,P-Erk1/2 and Collagen Ⅰ pro-tein expression on HSC. Conclusions CPhGs has the protective effect against hepatic fibrosis. The mecha-nism of this process may involve the interference with PDGF/ERK1/2 signaling pathway and inhibiting the activation and proliferation of HSC.

Effectiveness of the combinational treatment of Laminaria japonica and Cistanche tubulosa extracts in hair growth / 한국실험동물학회지

Since scalp hair loss has increased recently even in young people, seriously affecting individual's quality of life, the hair growth-stimulating effects of Laminaria japonica extract (LJE) and Cistanche tubulosa extract (CTE) were investigated. After confirming anagen phase of follicles under shaving, male C57BL/6 mice were dermally applied with 3% Minoxidil or orally administered with the combinations of LJE and CTE for 21 days. Minoxidil promoted the hair regrowth and increased gamma-glutamyl transpeptidase (gamma-GTP) and alkaline phosphatase (ALP) activities. In addition, Minoxidil up-regulated epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF) levels. Co-administration of LJE and CTE at 54 mg/kg LJE plus 162 mg/kg CTE exerted synergistic promoting effects on the hair regrowth, comparable to 3% Minoxidil. LJE preferentially enhanced ALP activity, while CTE increased both gamma-GTP and ALP activities as well as EGF and VEGF expressions. In vivo air pouch inflammation model, carrageenan-induced vascular exudation and increased nitric oxide and prostaglandin E2 concentrations in the exudates were synergistically suppressed by co-administration of LJE and CTE. In addition, inflammatory cell infiltration was substantially inhibited by the combinational treatment. The results suggest that combinational oral treatment with LJE and CTE in appropriate doses and ratios prevent hair loss and improve alopecia, which might be in part mediated by their anti-inflammatory activities.

Efficacy of Cistanche Tubulosa and Laminaria Japonica Extracts (MK-R7) Supplement in Preventing Patterned Hair Loss and Promoting Scalp Health

Cistanche tubulosa and Laminaria japonica have been reported to have anti-oxidative, anticoagulant, anti-cancer and anti-inflammatory properties. They are expected to be a promising candidates for promoting hair growth and treating dandruff and scalp inflammation as a consequence. In this double-blinded, placebo-controlled clinical trial, we investigated the efficacy of Cistanche tubulosa extract and Laminaria japonica extract complex (MK-R7) in promoting hair health in patients with mild to moderate patterned hair loss. Using phototrichogram (Folliscope 4.0, LeadM, Seoul, Korea), we compared the density and diameter of hairs in patients receiving a placebo or Cistanche tubulosa extract and Laminaria japonica extract complex (MK-R7) at baseline, 8 and 16 weeks of the study. In order to determine the efficacy of treatment on dandruff and scalp inflammation, investigator's assessment score and patient's subjective score were also performed. We found a statistically significant increase in the hair density of the test group (n = 45, MK-R7 400 mg) after 16 weeks of consuming the MK-R7 (test group: 23.29 n/cm2 +/- 24.26, control: 10.35 n/cm2 +/- 20.08, p < 0.05). In addition, we found a statistically significant increase in hair diameter in the test group compared to control group at week 16 (test group: 0.018 mm +/- 0.015, control: 0.003 mm +/- 0.013, p < 0.05). There were also significant outcomes regarding the investigator's visual assessment and patient's subjective score of dandruff and scalp inflammation in the test group compared to those in control group. Based on the results of this clinical study, we conclude that Cistanche tubulosa extract and Laminaria japonica extract complex (MK-R7) are promising substances for promoting health of the scalp and hair.

Comparison of DNA extraction methods from four Chinese traditional medical herbs / 中国生化药物杂志

Objective To establish an optimum DNA extraction method for Chinese traditional medical herbs in order to meet necessary for DNA barcoding research.Methods Four Chinese traditional herbs, Glycyrrhiza uralensis Fisch, Phellodendron Chinensis Cortex, Cistanche tubulosa Wight and Cistanche deserticola Ma were chosen as the experimental materials, the DNA was extracted by 6 different kinds of DNA extraction method, including the improved method of high-salt combined low-pH,the improved method of SDS,CTAB method,PVP method,PlantZol Kit and Ezup Kit, the quality of DNA was investigated by ultraviolet spectrophotometry,agarose gel electrophoresis and PCR amplification by using specific primers of ITS2 and psbA-trnH. ResuIts The quality of the DNA was better than other four kinds of methods by the improved method of high-salt combined low-pH and Ezup Kit, the value of OD260/OD280 was between 1.7 ~1.9,the yield of DNA was the highest by the PlantZol kit , followed by the improved method of high-salt combined low-pH( P <0.05 ) , but the purity of DNA was poor by the PlantZol kit.The DNA electrophoresis tests showed that the DNA integrity of Glycyrrhiza uralensis Fisch and Cistanche tubulosa Wight were better with the improved method of high-salt combined low-pH, the improved SDS method, the CTAB method and the PlantZol kit.The DNA of Phellodendron Chinensis Cortex and Cistanche deserticola Ma were extracted by the six methods appeared diffuse status in the lanes.But only the improved method of high-salt combined low-pH could make the PCR amplification of the success rate 100% by using specific primers of ITS2 and trnH-psbA.ConcIusion The DNA extraction method of high-salt combined low-pH can be used to establish the Chinese DNA barcoding which has the advantages of lower cost, simpler procedure and less time.

Extraction of phenylethanoid glycoside from Cistanche tubulosa and purification technology by macroporous resin / 中草药

Objective: To optimize the extraction and purification technology of phenylethanoid glycoside from Cistanche tubulosa. Methods: Orthogonal design L9(34) was employed to optimize the extraction conditions by taking the extraction yield of echinacoside and acteoside as indexes. The absorption-desorption characteristics of seven macroporous resins were evaluated, then the best purification conditions were optimized. Results: The optimal extraction conditions were as follows: The air-dried stems of C. tubulosa were powdered and extracted twice with 12-fold 50% ethanol for 1 h each time, temperature was 70℃; The supernatant was concentrated to 5-fold weight of the stems of C. tubulosa. The concentrated liquid was subjected to macroporous resin (HPD750) column and then eluted with deionezed water (3 BV) and 30% ethanol (4 BV), respectively. The 30% ethanol fraction was evaporated under vacuum to give the phenylethanoid glycoside-rich C. tubulosa stem extract. The purity of phenylethanoid glycoside was above 75%. Conclusion: The optimized extraction and purification process is stable, efficient, and suitable for industrial production.

Synergistic anti-inflammatory effects of Laminaria japonica fucoidan and Cistanche tubulosa extract / 한국실험동물학회지

The anti-inflammatory effects of fuciodan and Cistanche tubulosa (CT) extract were investigated in vitro macrophage culture system and in vivo carrageenan-induced air pouch inflammation model. CT extract inhibited nitric oxide production from activated RAW 264.7 macrophage cells, while fucoidan was inactive. In vivo air pouch inflammation model, carrageenan-induced vascular exudation and increased nitric oxide and prostaglandin E2 concentrations in the exudates were synergistically suppressed by co-administration of fucoidan or CT extract. Moreover, tissue inflammation was substantially attenuated by the combinational therapy. However, there was no synergistic effect against the inflammatory cell infiltration, although fucoidan and CT extract each markedly reduced the cell numbers. Therefore, it is suggested that fucoidan blocks infiltration of inflammatory cells, while CT extract inhibits activation of the cells, and that their combinational treatment could be a promising candidate for the relief of various types of inflammation.

Chemical constituents in artificial cultivated Cistanche tubulosa / 中草药

Object To analyze the chemical constituents and their variation at different growing time and segment in artificial cultivated Cistanche tubulosa(Schrenk) Wight. Methods Using HPLC to analyze the fingerprint spectrum of different samples. Results The sorts of phenylethanoid glycosides were almost the same among different growing time, different segments, cultivated and wild species, and every parts of C. tubulosain blooming, but the contents of these chemical constituents are significantly different among various samples. Conclusion The cultivating time of C. tubulosashould be over three-years, its harvest time must be controlled strictly before blooming. The quality of cultivated plants is no better than wild ones still. So the cultivating technology should be promoted to improve the content of active compounds.

Fingerprints of active principles in Cistanche tubulosa from different regions / 中成药

AIM: A HPLC fingerprint has been established of six active principles in Cistanche tubulosa in nine different regions in the Xinjian Uygur Automomous Region in China. METHODS: Chromatographic condition included a symmetry C_(18)(4.6 mm?250 mm,5 ?m) column and the gradient elution was adopted with ratio of acetonitrile-0.095% H_3PO_4 from 8()∶92 to 12()∶88 in 0-18 min and 12()∶18 to 19()∶81 in 18-40 min,19()∶81 maintain for 50 min.The detection wavelength was at 330 nm and the flow rate was 1.0 mL/min and detection time was 90 min. RESULTS: Eleven common characteristic peaks,including salidroside, bartsioside,echinacoside,cistanbuloside A,tabuloside A and acteoside were taken as fingerprint peaks the precision and the accuracy were in accordance with the chromatographic requirement. CONCLUSION: The method is stable,reliable,precision and provides a scientific basis for the quality standard for Cistanche tubulosa.