Differential Constituents in Roots, Stems and Leaves of Polygonum multiflorum Thunb. Screened by UPLC/ESI-Q-TOF-MS and Multivariate Statistical Analysis.

The differential constituents in leaves, stems and roots of Polygonum multiflorum Thunb. were analyzed by ultra-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry (UPLC/ESI-Q-TOF-MS) and by multivariate statistical analysis. The established extraction and analysis method showed relative standard deviations (RSDs) for intra-day precision of less than 3.40%, for repeatability of less than 4.06% and for stability of less than 5.10%. Principal component analysis and orthogonal projections to latent structures discriminant analysis of the UPLC/ESI-Q-TOF-MS data showed good ability to classify the leaves, stems and roots of P. multiflorum Thunb. The differential constituents, such as stilbenes, polygoacetophenoside, flavonoids and anthraquinones, accounting for variations between the leaves, stems and roots, were filtered through the variable importance in projection values and were further identified by elemental composition analysis, mass fragmentation data and retention times of available standards. Differences between the chemical compositions in the leaves, stems and roots of P. multiflorum Thunb. were closely related to their various therapeutic effects. This UPLC/ESI-Q-TOF-MS-based analytical strategy could be further utilized to evaluate the overall quality of traditional Chinese medicines and their differences of chemical constituents in different parts of the plant and/or in the plants of different geographical locations.

Species identification of Chinese medicinal plant Fallopia multiflora (Thunb.) Haraldson by suppression subtraction hybridization.

Fallopia multiflora (Thunb.) Haraldson, a traditional Chinese medicinal plant, has been extensively used in preparations of herbal medicine, health products and personal hygiene products. However, the clinical safety and efficiency of F. multiflora (Thunb.) Haraldson is impaired because of the existence of various adulterants. In this study, genomic DNA (gDNA) suppression subtraction hybridization (SSH) was used to authenticate F. multiflora (Thunb.) Haraldson from its adulterants. First, differential gDNA fragments between F. multiflora (Thunb.) Haraldson and its most closely related species F. multiflora var. ciliinervis (Nakai) Yonek. & H. Ohashi by SSH were identified. The differential fragments were then hybridized with arrays constructed from multiple whole genomes of several species (adulterants and/or closely related plants) to screen for the unique gDNA fragments representing F. multiflora (Thunb.) Haraldson. The unique gDNA fragments could be used to design species-specific primers for the identification of F. multiflora (Thunb.) Haraldson. Using SSH, we obtained four differential gDNA fragments, and four pairs of primers were designed. The designed primers could differentiate F. multiflora (Thunb.) Haraldson from its adulterants and/or closely related species via PCR. The results confirmed that the SSH is an efficient method for screening and designing species-specific primers.

[Molecular authentication of Fallopia multiflora by PCR-RFLP based on the trnL-trnF analysis].

OBJECTIVE: To establish a method for the molecular authentication of Fallopia multiflora. METHODS: The trnL-trnF regions of Fallopia multiflora and its closely related species and/or adulterants were sequenced and analyzed. RESULTS: It was found that the trnL-trnF sequence divergences between Fallopia multiflora and its closely related species and/or adulterants were 2.1%-22%. While the intra-species trnL-trnF divergences of Fallopia multiflora were 0%-1.5%. Based on the trnL-trnF regional variations, an endonuclease Xba I (T CTAGA) restriction site specific to Fallopia multiflora was detected. The Fallopia multiflora trnL-F polymerase chain reaction product could be cleaved by Xba I into two pieces, 804-819 bp and 256 bp each, whereas the restriction endonuclease could not digest the trnL-trnF polymerase chain reaction product of its closely related species or adulterants. The restriction patterns analyzed for restriction enzyme Xba I were found to be identical in all Fallopia multiflora individuals from different geographical regions in China. CONCLUSION: The assay based on polymerase chain reaction amplification of the trnL-trnF fragment of chloroplast DNA and subsequent restriction fragment length polymorphism can be used as a general test to identify Fallopia multiflora.

An effective method of RNA isolation from Fallopia multiflora tuberous roots.

To isolate high-quality total RNA from Fallopia multiflora tuberous roots is difficult because of the presence of high levels of carbohydrates, phenolics, and other secondary metabolites. Since several procedures specialized for RNA isolation from polysaccharides and phenols rich tissues have resulted in poor yields, in this study, we developed a modified protocol that was derived from the traditional CTAB method. The protocol was able to produce high-quality and intact RNA from the tuberous roots of F. multiflora. The yield of total RNA was more than 0.15 mg/g fresh weight, with an A260/A280 ratio of 1.9-2.0. The obtained RNA was of sufficient quality and suitable for downstream application such as reverse-transcription polymerase chain reaction (RT-PCR), Northern hybridization, and cDNA library construction. The protocol may also have wider applicability for total RNA isolation from other plant species with tuberous roots.

[Influence of najanalgesin from Naja naja on GLT-1 in spinal cord of rat in neuropathic pain].

OBJECTIVE: To investigate the influence of najanalgesin on glutamate-glial transporter 1(GLT-1) in spinal cord of rats after L5 spinal nerve ligation and transection (SNL), and explore the spinal analgesic mechanism of najanalgesin. METHOD: One hundred male SD rats were randomly divided into 6 groups: sham(A), SNL(B), SNL + najanalgesin(C), SNL + saline (D), SNL + najanalgesin + liposome (E), SNL + najanalgesin + liposome + GLT-1 As-ODNs(F) and treated with intrathecal injections of 10 p.L saline (A and D), 40 ng X kg(-1) najanalgesin (C, E and F), qd, respectively. Besides intrathecal administration of najanalgesin the rats were intrathecally injected with 10 microL of GLT-1 antisense oligodeoxynucleotides (As-ODNs) (F) and 10 micdroL of liposome(E) once daily on day 3. The L4-L6 segments of the spinal cord were isolated in 1, 4 and 7 d(A,B,C and D), 7 d(E and F) after surgery. The mRNA and protein of GLT-1 were determined. RESULT: The SNL model has successfully been set up. Compared to sham group, the expression of GLT-1 mRNA and protein level in group B and D both increased firstly and decreased later, the expression of GLT-1 in group C was significantly increased and kept stable, which were also higher when compared to group D in day 7th. Compared to SNL + najanalgesin group, after intrathecal injection of GLT-1 As-ODNs the GLT-1, expression of GLT-1 in F group significantly decreased. While intrathecal administration of liposome had no significant effect on the spinal GLT-1 expression. CONCLUSION: Najanalgesin could increase the mRNA and protein expression of GLT-1 in spinal cord, which may be one of its spinal mechanisms of analgesia.

[Analysis and authentication of Fallopia multiflora and its adulterants by matK sequence].

OBJECTIVE: To identify Fallopia multiflora from its adulterants by comparing their matK sequences. METHODS: Genomic DNA of different materials was extracted using modified cetytrimethyl ammonium bromide (CTAB) method. The double-strand matK genes were amplified using PCR method and then sequenced. The data were analyzed in Clustral W and MEGA 4.0 software package. RESULTS: Besides F. multiflora var. ciliinerve, the matK sequences of other adulterants show distinct differences with F. multiflora, whether for nucleotides substitutions or genetic distances; and the specific identifying sites for distinguishing F. multiflora and other Fallopia adulterants were found through further comparative analysis. Moreover, the 3 inspected materials were successfully authenticated by comparing the matK sequences. CONCLUSION: matK sequences can be used for the molecular identification between F. multiflora and its adulterant species.

[Isolation and pharmacological properties of analgesic fraction from venom of Naja Naja atra].

OBJECTIVE: To separate main analgesic fraction from venom of Guangdong Naja naja atra, to establish the basis for the using of Naja naja atra and find new analgesic fraction. METHODS: Affinity chromatography and size exclusion were used to isolate the analgesic fraction from the venom of Naja naja atra, and then to determine its properties by biochemical methods, such as SDS-polyacryamide gel electrophoresis ( SDS-PAGE), HPLC and Mole-toff. RESULTS: HPLC showed its relative purity was 95% (HPLC)and Mw was 6741. 236 Da. We observed that peripheral administration of neurotoxin strongly reduced the mechanical allogynia and thermal hyperalgesia for 24 hours, associated with this neuropathy (L5 spinal nerve ligation). CONCLUSION: The fraction from venom of Naja naja atra has significant analgesic effect and it is worth further developing.

Molecular authentication of the traditional medicinal plant Fallopia multiflora.

The root of Fallopia multiflora is one of the most widely used traditional Chinese medicines. However, it is often confused and substituted with the roots of F. multiflora var. ciliinervis, Pteroxygonum giraldii, Cynanchum auriculatum, and Stephania cepharantha. To establish a DNA polymorphism-based assay for the identification of F. multiflora, the nuclear ribosomal DNA (nrDNA) internal transcribed spacer (ITS) regions of six Fallopia species were sequenced and analyzed. Based on the diversity of ITS regions among the species the diagnostic primers PMITS28 and PMITS545, which amplified an expected 517-bp DNA fragment from F. multiflora DNA, were designed. No amplified product was observed when DNA from other species was used. This method can be used for the authentication of F. multiflora.

Genetic variation and identification of cultivated Fallopia multiflora and its wild relatives by using chloroplast matK and 18S rRNA gene sequences.

FALLOPIA MULTIFLORA (Thunb.) Harald . has been widely and discriminatingly used in China for the study and treatment of anemia, swirl, deobstruent, pyrosis, insomnia, amnesia, atheroma and also for regulating immune functions. However, there is still confusion about the herbal drug's botanical origins and the phylogenetic relationship between the cultivars and the wild relatives. In order to develop an efficient method for identification, a molecular analysis was performed based on 18 S rRNA gene and partial MATK gene sequences. The 18 S rRNA gene sequences of F. MULTIFLORA were 1809 bp in length and were highly conserved, indicating that the cultivars and the wild F. MULTIFLORA have the same botanical origin. Based on our 18 S rRNA gene sequences analysis, F. MULTIFLORA could be easily distinguished at the DNA level from adulterants and some herbs with similar components. The MATK gene partial sequences were found to span 1271 bp. The phylogenetic relation of F. MULTIFLORA based on the MATK gene showed that all samples in this paper were divided into four clades. The sequences of the partial MATK gene had many permutations, which were related to the geographical distributions of the samples. MATK gene sequences provided valuable information for the identification of F. MULTIFLORA. New taxonomic information could be obtained to authenticate the botanical origin of the F. MULTIFLORA, the species and the medicines made of it.

[Radioprotection related activities of medicinal plant].

The development of radioprotective agents has been the subject of intense research in view of their potential for use within a radiation environment, such as space exploration, radiotherapy and even nuclear war. However, no ideal synthetic radioprotectors are available at present, so the search for alternative sources, including plants, has been on going for several decades. This article reviews some of the most promising plants, and their radioprotection related activities.

[Immunoloregulation effect of polysaccharide isolated from octopus dollfusi on mouse].

OBJECTIVE: To study the immunoloregulation effect of three polysaccharides OGI, OG2and OG3 extracted from Octopus dollfusi muscle, gonad, digestive gland, respectively. METHODS: Spleen cell proliferation was measured by MTT assay and index of immune organs were weighed and calculated. RESULTS: OGI and OG2 could increase the proliferation of mouse spleen cell of normal mice, and significantly increased the proliferation of the spleen of immunosuppression mice caused by sccyclophosphamide, while showed no cooperation with ConA; OG3 appeared suppression for the two spleens. The three polysaccharides could increase index of immune organs of normal mice and remarkably increased the indexof immunosuppression mice caused by sccyclophosphamide ; while showed obvious function on thymus index. CONCLUSION: These results suggest that OG1 and OG2 have enhancement immunity for normal and immunosuppression mice, and have better effect for the latter;OG3 has suppression proliferation the spleen cell in vitro, however it has better effect for increase index of immune organs in vivo.

[Comparison of content of 2,3,5,4'-tetrahydroxystilbene-2-O-beta-D-glucoside and anthraquinon in different ploidy Radix Polygoni Multiflori].

OBJECTIVE: To investigate the content of 2,3,5,4'-tetrahydroxystilbene-2-O-beta-D-glucoside and anthraquinon in diploid and triploid Radix Polygoni Multiflori. METHODS: 4 batches of Radix Polygoni Multiflori were collected from different districts. The content of 2,3,5,4'-tetrahydroxystilbene-2-O-beta-D-glucoside and anthraquinon in these samples were determined at 320 nm and 254nm wave length by HPLC with Inertsil ODS-3 C18 pillar and acetomitrile: aqua (25:75), methanol: 0.1% phosphoric (85:15) respectively as the mobile phase. RESULTS: The maximum content of 2,3,5,4'-tetrahydroxystilbene-2-0-beta-D-glucoside was diploid Radix Polygoni Multiflori from Deqing Guangdong. The maximum ratio of total anthraquinon was triploid Radix Polygoni Multiflori from Jinxi Guangxi reached 85%. CONCLUSION: The content of anthraquinon varies greatly in the samples from the different producing areas.

[Extraction and purification of psoralen from Psoralea corylifolia L].

OBJECTIVE: To establish a method for extracting psoralen from the seed of Psoralea corylifolia L. METHODS: Crushed seed of Psoralea corylifolia L. was soaked with 50% ethyl alcohol solution, and the leaching solution was filtrated and distilled to remove alcohol. A slurry of the sediment was obtained and dissolved in methyl alcohol (MeOH), followed by treatment with active carbon for decoloration and evaporation of MeOH, resulting in a lamellar crystal after the sediment was kept still overnight. MeOH was applied to dilute the sticky solution, and after filteration, a yellow crystal was obtained, recrystallization of which in MeOH yielded white needle-like crystals. Thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) were employed to determine the purity of the resultant substance, and UV and H1-NMR applied to identify its chemical structure. RESULTS AND CONCLUSION: The resultant crystal is confirmed to be pure monomers of psoraren with a yield of 0.147% from the seed (m/m) by this simple extraction method.

[Purification of chymopapain and its effect on nucleus pulposus in vitro].

OBJECTIVE: To establish a simple and effective procedure for purification of chymopapain and study about its effect on nucleus pulposus in vitro. METHODS: Chymopapain was purified by ion exchange chromatograph and tested its effect on nucleus pulposus in vitro. RESULTS: A simple and effective procedure for purification of chymopapain was established and the chymopapain did degrade nucleus pulposus. CONCLUSION: The ion exchange chromatograph was a simple and effective procedure for purification of chymopapain. It is necessary to study its effect on nucleus pulposus in vivo.