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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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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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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 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 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.
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Object The polysaccharide (CDPS) was isolated, purified and identified from Cistanche deserticola Y. C. Ma, a Chinese materia medica. Methods The polysaccharide was extracted with hot water and precipitated by alcohol. Protein in the precipitates was removed by Sevag method. The products were further purified with column chromatography on DEAE-Sephadex A-50 and Sephacryl S-200. The CDPS was idendified by IR spectrum and UV (200-400 nm) scanning spectrum. Results IR spectrum indicates that there are typical characteristic absorption peaks of polysaccharides. UV scanning spectrum shows that there are no absorption peaks of protein and nucleic acid at point 280 nm and 260 nm. Conclusion The CDPS was identified as homogeneous one.
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Objective To improve the 2,3,5-triphenyl tetrazolium chloride (TTC) solution method for measuring the viability of Cistanche deserticola seeds and investigate the change in viability during storage at 5 ℃. Methods The effect of the testa,TTC concentration,sodium hypochlorite concentration (NaClO),and staining time were studied,and seed viability during storage at 5 ℃ was measured with the improved method. Results Seeds were kept for 48 h in 0.5% TTC solution at 40 ℃,and then for 2 h in 0.2% NaClO solution;Seed viability was measured under a stereomicroscope. Storing seeds of C. deserticola for 1 to 2 years at 5 ℃ had no significant effects on their viability. However,the percentage of seeds with high viability was increased with the extension of the storage time at 5 ℃. Conclusion A convenient and rapid method for measuring the viability of C. deserticola seeds is developed. Storing C. deserticola seeds at 5 ℃ could improve their viability
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Objective To determine the genetic diversity of germplasm resource in cultivated and wild Cistanche deserticola. Methods Fifty-eight samples from three populations of cultivated and wild C. deserticola were analyzed by amplified fragment length polymorphism (AFLP) DNA markers, and the gene- tic diversity was evaluated by PopGen32. Results The average percentage of polymorphic loci (PPL) of cultivated C. deserticola is 79.16%. The PPL of wild population is 89.53%. Average Neis gene diversity index (He) from four populations was 0.193 8, Shannons genetic diversity index (I) was 0.300 4, and genetic differentiation index (Gst) was 0.097 9. Conclusion The diversities of cultivated and wild C. deserticola are both higher and theres no differentiation between them. It shows that genetic diversity of inner-species is higher, which is not the reason for endangerment. Therefore, wild nursery and artificial cultivating are the best measures for the conservation and sustainable utilization in C.deserticola.
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Object The process of seed germination and haustorium formation in Cistanche deserticola Y C Ma was observed Methods Seeds were inoculated on culture medium, the process of seed germination and haustorium formation were observed using light and electron microscopy Results The seedling sprouted after two weeks, then a tube like organ formed, finally the apex expanded to attachment organ The outer papillar surface of the extended apex bears wall protuberances that are encircled with a thick cuticular belt and covered with a thin cuticle Conclusion The seed germination of C deserticola is single pole of racidle The attachment organ adheres host first
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Objective To establish chromatographic fingerprint of Cistanche deserticola by HPLC and evaluate the difference of inner qualities of samples from different habitats.Methods HPLC gradient elution was applied to establish the chromatographic fingerprint and "Computer Aimed Similarity Evaluation System" was used in data analysis.Results This chromatographic fingerprint method has good precision, stability, and repeatability; the fingerprints of the samples from different habitats were quite different.Conclusion There are notable differences in inner qualities of the samples from different habitats.This chromatographic fingerprint method can be used to evaluate the quality of C.deserticola.
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AIM: To study the protective effect of ci stanche deserticola Y. C Ma. on thymocytes in septic rats. METHODS: Cecum ligation perforation (CLP) was used to induce sep sis. Treatment group was treated with cistanche deserticola Y. C Ma. (1.25 g?kg -1?d -1, ig) for 14 days before CLP. Animals were killed 12 h or 24 h after CLP an d thmocyte s were collected. The ratio of thmocyte apoptosis and mitochondrial membrane pot ential were determined by the flow cytometry. The ATP activity was detected by s pectrophotography. RESULTS: The rate of thmocyte apoptosis significantly increased 12 h after CLP. The ATP activity decreased 24 h after CLP was significant. The extract of desert living cistanche effectively repressed the apoptosis of th ymocytes and maintained the mitochondrial membrane potential. CONCLUSIONS: The extract of cistanche deserticola Y. C Ma. p rotects thymocytes against apoptosis induced by sepsis. Maintaining of mitochond rial membrane potential may be the protective mechanism.