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In this study, according to TCM theory of "liver qi stagnation forming fire", emotional stress mice model was employed to evaluate the protective effects of Qingre Xiaoyanning on herpes simplex virus type 1 (HSV-1) induced reactivation. The animal experimental protocol has been reviewed and approved by Laboratory Animal Ethics Committee of Jinan University, in compliance with the Institutional Animal Care Guidelines. BALB/c mice were divided into six groups, including mock group, HSV-1 latency group, HSV-1 reactivation group (HSV-1 latency + stress), low (0.658 g·kg-1·day-1) and high dose (1.316 g·kg-1·day-1) of Qingre Xiaoyanning groups and positive control group (acyclovir, 0.206 g·kg-1·day-1). Except for the normal group and HSV-1 latency group, all mice in other groups received a daily 12-h restraint stress for 4 days. After 7-day treatment of drugs, body weight and recurrent eye infections of mice were recorded. Brain tissues were harvested to monitor HSV-1 antigen distribution by immunohistochemical staining and detect virus titer by plaque assay. In the meantime, the mRNA and protein levels of infected cell polypeptide (ICP27) and glycoprotein B (gB) in the brain tissues were detected by RT-PCR and Western blot, respectively. The level of 4-hydroxynonenal (4-HNE) and expressions of ferroptosis-related proteins were measured by Western blot. The evaluation of malondialdehyde (MDA) content in the brain tissues was conducted by MDA assay commercial kit. The results showed that Qingre Xiaoyanning significantly retarded the decline of body weight of mice induced by HSV-1 reactivation, reduced the activation rate of HSV-1 and recurrent eye infections, declined virus titer of HSV-1, down-regulated gene and protein expressions of ICP27 and gB, and hindered the distribution of HSV-1 antigen in the brain of mice. Meanwhile, Qingre Xiaoyanning also decreased the protein expression of ferroptosis-related proteins, including DMT1, TFR1 and ALOX15 in the brain tissue of HSV-1 reactivated mice. The levels of lipid peroxidation products, 4-HNE and MDA, were also reduced by Qingre Xiaoyanning treatment. All the above results indicate that Qingre Xiaoyanning significantly inhibited HSV-1 reactivation by restraint stress, which might be related to the regulation of ferroptosis. Our findings provide a theoretical basis for the application of "clearing liver-fire" TCM on treatmenting HSV-1 reactivation-related symptoms.
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Objective: To identify Sarcandra glabra and its adulterants using three DNA molecular markers including 18 S rRNA gene, ITS2 sequence and SCAR marker, and then provide the basis for its molecular authentication. Methods: 18 S rRNA gene sequence of S. glabra was obtained by PCR amplification, cloning and sequencing, and then Blast comparison was made in NCBI. The ITS2 sequence of S. glabra was obtained by PCR amplification, sequencing and annotation in ITS2 Database. In the meanwhile, the ITS2 sequences of adulterants and other plants were collected from GenBank. Using MEGA5.5, the genetic distance was calculated between species and then the ITS2 sequences were aligned to construct a phylogenetic clustering tree. SCAR molecular marker of S. glabra was obtained by RAPD. After cloning and sequencing, specific primers were designed to amplify S. glabra and its adulterants. Results: The length of 18 S rRNA obtained in our research was 1 820 bp. Blast comparison revealed that there was 99% homology between S. glabra and Chloranthaceae, which proved to be 18 S rRNA gene of S. glabra. The length of the ITS2 sequence in our research was 500 bp. Genetic distance between S.glabra and its adulterants ranged from 0.190 to 0.219, which was far more than genetic distance among adulterants (0.000-0.074). Cluster analysis showed that S. glabra and its adulterants respectively clustered into a different branch, which was far away from other plants. In our research, we obtained SCAR molecular marker of S. glabra and then a pair of specific primers were designed. Using the pair of specific primers, specific products were amplified from genomic DNA of S. glabra, but no specific products were obtained from that of its adulterants. Conclusion: We could authenticate S. glabra and its adulterants effectively with the combination of three molecular markers for establishing a novel method to identify S. glabra and its adulterants, which provides a new idea for the authentication of S. glabra.
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Objective: Sarcandra glabra was recognized as an important research material attributing to its high medicinal value and economic value. However, little information was known about its genomics and regulatory pathway participating in reproductive development. For the first step to understand the molecular basis and further explore genes which related to metabolism and resistance in S. glabra. Methods: A SMART full-length complementary DNA library from the leaves tissue was constructed and characterized to providing the experimental basis for discovery of functional genes of S. glabra. The assembly expressed sequence tag (EST) data were completed by ABI3730 DNA program. A high quality full-length cDNA library was constructed successfully from S. glabra leaves. Results: The titer of library was 1.14×107 pfu/mL and the average length of inserted fragments was 1 000 bp. A total of 221 clones were sequenced from the cDNA library and obtained 177 EST sequences. The EST sequences were assembled into 151 unigenes including 12 contigs and 119 singletons (79%). EST exhibited significant similarity with known putative functional nucleotide sequences in the GenBank database. These genes were mostly involved in cell development, signal transduction, protein synthesis, transcription, stress tolerance response, energy metabolism based on molecular function of GO annotation. Conclusion: This report constructs a full-length-cDNA library and analyzes the bioinformatics of the related EST sequences, and then offers a reference to genomic research of S. glabra.
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Objective: To study the ribosomal DNA internal transcribed spacer (rDNA-ITS) sequences of Sarcandra glabra from different areas and five plants in Chloranthus Swartz, and to provide pattern recognition and thread for the species identification of S. glabra. Methods: The ITS sequences of 18 populations of S. glabra and six populations of Chloranthus spicatus were amplified by PCR with universal primer of ITS and sequenced, and the ITS sequences of the other plants in Chloranthus Swartz were searched in GeneBank. Data were analyzed by ClustalX 2.1, and a cluster analysis was presented by UPGMA. Results: The semblance of ITS sequences of S. glabra from different areas was 99%. The total mutation rate of ITS1 sequence (2.7%) was higher than that of ITS2 sequence (1.4%). However, compared with other plants in Chloranthus Swartz, the total mutation rate of ITS2 sequence (20.3%-22.7%) was higher than that of ITS1 sequence (15.9%-18.3%). The cluster analysis showed that there was little variation among the 18 populations of S. glabra, but there was significant difference with the five plants of Chloranthus Swartz. Conclusion: ITS sequence can be used to identify the plants from different areas and in different genus, ITS sequence of S. glabra has several specified information sites to identify the five plants in Chloranthus Swartz, with significantly different cluster analyseis, and is the active molecular marker for the species identification of S. glabra and plants in Chloranthus Swartz.
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Objective: To analyze the correlation of DNA fingerprints of Sarcandra glabra with different quality levels and its quality. Methods: Using ISSR-PCR, 100 ISSR primers (UBC801-UBC900) were screened, and 23 of them were polymorphic. The 23 ISSR primers were used to amplify 18 S. glabra samples from different habitats. Based on the 18 ISSR amplified bands, the data base of amplified bands fingerprint was established using Excel. Results: one hundred and ninty-eight bands were obtained. Each primer amplified eight bands on average. The number of polymorphic DNA bands was 184, and the polymorphic proportion of DNA bands was 92.9%. Seven sites in two primers (UBC811 and UBC825) screened from 23 polymorphic ISSR primers were in one group, and seven sites in three primers (UBC827, UBC834, and UBC842) were in another group. The DNA fingerprints of 18 S. glabra samples were established, and U844-6 bands were screened from 184 polymorphic bands to correlate with sample quality. Conclusion: ISSR molecular markers could identify the DNA fingerprints of 18 S. glabra samples, and screen one band that is related to the quality.
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Objective To study the effects of drought stress on chlorophyll fluorescence characteristics and energy dissipation of Sarcandra glabra and get the method of alleviating drought stress, so as to provide theoretical basis against drought in planting. Methods Several physiological indexes of S. glabra treated by 5-aminolevulinic acid (ALA) under drought stress (PEG-6000 at the concentration of 15%) were measured, such as the contents of photosynthetic pigment, chlorophyll fluorescence parameters, and energy parameters. Results Exogenous ALA obviously increased the content of chlorophyll and carotenoids, enhanced the maximum fluorescence (Fm), photochemical efficiency of photosystem II (PSII, Fv/Fm), potential photochemical efficiency (FWFo), photochemical efficiency (Fv'/Fm'), PSII actual photochemical efficiency (φPSII), photochemical quenching coefficient (qP), electronic transfer rate (ETR), and photochemistry rate (PCR), as well as significnatly decreased the level of minimum fluorescence (Fo), non-photochemical quenching (NPQ) coefficient, and heat dissipation rate (HDR). The proportion of ALA absorbed light in photochemistry (P) was increased, the fraction of antenna pigment heat dissipation (D) and excess energy (E) for NPQ was decreased. The fraction off was the main pathway for excessive energy dissipation. ALA could promote the redistribution of energy reasonablely. Conclusion Exogenous ALA (100 mg/L) could significantly reduce the dissipation of excess excitation energy, improve the photochemical electron transport efficiency, and efficiently protect leaf blade of S. glabra from PSII damage under drought stress. ALA could obviously promote the drought resistance of S. glabra plantlet.
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Objective Sarcandra glabra is a widely-used Chinese medicinal herb,but the wild germplasm resources were decreasing.In order to protect the germplasm resources,the genetic diversity and genetic relationship among the various provenances should be analyzed.Methods ISSR Molecular markers were used to analyze the genetic diversity of S.glabra collected from eight provenances.The genetic diversity,genetic distance,and cluster analysis were performed by Popgen32 software and UPCMA method.Results Ten screened polymorphic primers were used in ISSR-PCR and 111 bands were amplified,among them including 106 polymorphic bands that were about 95.50%.Genetic distance of eight different provenances were ranged from 0.034 7—0.185 0.The genetic diversity of S.glabra mainly came from the interior variation of every provenance,for Gst=0.340 3.Conclusion Provenances of S.glabra can be divided into two groups and based on the relationship of genetic distance and altitude,the provenances can be divieded as follows:correspondingly higher altitude group(800-900 m);correspondingly lower altitude group(250-300 m);middling altitude group(600 m).
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Objective To set up the technology for adsorbing and separating total flavone in Sarcandra glabra with macroporous adsorption resin,11 types of macroporous adsorption resins were investigated.Methods The static adsorption and separation were used in investigation of macroporous adsorption resin;the dynamic adsorption and separation were used in studying the condition of adsorption and separation.The total flavone adsorption capacity,total flavone content,and total flavone recovery rate were used as the evaluation criteria.The UV spectrophotometric method was used in the determination of total flavone content.Results The results showed that among 11 types of macroporous adsorption resins,the HPD400 was the best for adsorbing and separating the total flavone in S.glabra in the following technological condition: the concentration of S.glabra sample extract was 10 mg/mL;the maximum adsorbing capacity for total flavone in S.glabra was 9.5 mg/mL;the current velocity was 2.5 mL/min;the eluting reagent was 70% ethanol(three times as the volume of the resin);and the HPD400 resin could be used three times,repeatedly.Conclusion It is a simple and efficient to separate the total flavone in S.glabra under the technological conditions,and a total flavone recovery rate is about 85%.