[One new coumarin from seeds of Herpetospermum pedunculosum].

This paper aims to investigate the chemical constituents of the seeds of Herpetospermum pedunculosum. One new coumarin and two known lignans were isolated from the ethanolic extract of the seeds of H. pedunculosum with thin layer chromatography(TLC), silica gel column chromatography, Sephedax LH-20 chromatography, Semi-preparative high performance liquid chromatography and recrystallization, etc. Their structures were elucidated as herpetolide H(1), phyllanglaucin B(2), and buddlenol E(3) by analysis of their physicochemical properties and spectral data. Among them, compound 1 was a new compound, and compounds 2 and 3 were isolated from this genus for the first time. In vitro anti-inflammatory activity test showed that herpetolide H had certain NO inhibitory activity for LPS-induced RAW 264.7 cells, with its IC_(50) value of(46.57±3.28) µmol·L~(-1).

[New records of medicinal plants in Tibet].

By the fourth survey of Chinese medicinal resources, new medicinal plants records of 2 genera and 5 species were reported in Tibet. They are two genera Rhynchoglossum and Asteropyrum, and five species including Rh. obliquum, A. peltatum, Urena repanda, Schefflera khasiana and Mimulus tenellus. All the voucher specimens are preserved in Herbarium of Tibet Agriculture and Animal Husbandry University.

Integrated analysis of transcriptomic and metabolomic data reveals critical metabolic pathways involved in rotenoid biosynthesis in the medicinal plant Mirabilis himalaica.

Mirabilis himalaica (Edgew.) Heimerl is among the most important genuine medicinal plants in Tibet. However, the biosynthesis mechanisms of the active compounds in this species are unclear, severely limiting its application. To clarify the molecular biosynthesis mechanism of the key representative active compounds, specifically rotenoid, which is of special medicinal value for M. himalaica, RNA sequencing and TOF-MS technologies were used to construct transcriptomic and metabolomic libraries from the roots, stems, and leaves of M. himalaica plants collected from their natural habitat. As a result, each of the transcriptomic libraries from the different tissues was sequenced, generating more than 10 Gb of clean data ultimately assembled into 147,142 unigenes. In the three tissues, metabolomic analysis identified 522 candidate compounds, of which 170 metabolites involved in 114 metabolic pathways were mapped to the KEGG. Of these genes, 61 encoding enzymes were identified to function at key steps of the pathways related to rotenoid biosynthesis, where 14 intermediate metabolites were also located. An integrated analysis of metabolic and transcriptomic data revealed that most of the intermediate metabolites and enzymes related to rotenoid biosynthesis were synthesized in the roots, stems and leaves of M. himalaica, which suggested that the use of non-medicinal tissues to extract compounds was feasible. In addition, the CHS and CHI genes were found to play important roles in rotenoid biosynthesis, especially, since CHS might be an important rate-limiting enzyme. This study provides a hypothetical basis for the screening of new active metabolites and the metabolic engineering of rotenoid in M. himalaica.

[Molecular cloning and characterization of CMK from Artemisia annua].

Artemisinin is a preferred medicine in the treatment of malaria. In this study, AaCMK, a key gene involved in the upstream pathway of artemisinin biosynthesis, was cloned and characterized from Artemisia annua for the first time. The full-length cDNA of AaCMK was 1 462 bp and contained an ORF of 1 197 bp that encoded a 399-anomo-acid polypeptide. Tissue expression pattern analysis showed that AaCMK was expressed in leaves, flowers, roots and stems, but with higher expression level in glandular secretory trichomes. In addition, the expression of AaCMK was markedly increased after MeJA treatment. Subcellular localization showed that the protein encoded by AaCMK was localized in chloroplast. Overexpression of AaCMK in Arabidopsis increased the contents of chlorophyll a, chlorophyll b and carotenoids. These results suggest that AaCMK plays an important role in the biosynthesis of terpenoids in A. annua and this research provids a candidate gene that could be used for engineering the artemisinin biosynthesis.

[Cloning and characterization of an aromatic amino acid aminotransferase(ArAT) gene involved in tropane alkaloid biosynthesis from Hyoscyamus niger].

Tropane alkaloids are anticholinergic drugs widely used clinically. Biosynthesis of tropane alkaloids in planta involves a step of transamination of phenylalanine. Based on the sequenced transcriptomes of lateral roots and leaves of Hyoscyamus niger, we found three annotated aromatic amino acid aminotransferases, which were respectively named HnArAT1, HnArAT2 and HnArAT3. Sequence analysis showed that HnArAT3 had highest similarity with the reported Atropa belladonna Ab Ar AT4, which was involved in tropane alkaloid(TA) to provide the precursor of the phenyllactic acid moiety. Tissue expression pattern analysis indicated that HnArAT3 was specifically expressed in lateral roots, where is the organ synthesizing tropane alkaloids. Then, method of virus induced gene silencing (VIGS) was used to characterize the function of HnArAT3 in H. niger. Gene expression analysis given by real-time quantitative PCR showed that all the transgenic lines had lower expression levels of HnArAT3 than the non-transgenic control, and HPLC analysis of alkaloids demonstrated significant decrease in the contents of hyoscyamine, anisodamine and scopolamine in planta. These results suggested that HnArAT3 was involved in the phenyllactic acid branch of TA biosynthetic pathway. Molecular cloning and functional identification of HnArAT3 laid the foundation for further understanding of TA biosynthesis and metabolic regulation, and also provided a new candidate gene for engineering biosynthetic pathway of tropane alkaloids.

Rhobupcyanoside B, A new cyanoside from Rhodiola bupleuroides.

One new cyanoside, rhobupcyanoside B (1), together with 7 known ones, was isolated from the 70% ethanol extract of the roots and rhizomes of Rhodiola bupleuroides. Their structures were determined by spectroscopic methods, including 2D NMR techniques. Compound 1 was evaluated for its inhibitory activity against α-glucosidase with IC50 value of 278.28 ± 0.55 µM by comparing with the positive control (acarbose) at 210.40 ± 0.32 µM.

[Chamerilactone A: one new isocoumarin isolated from Chamerion angustifolium].

A new isocoumarin, chamerilactone A(1) was isolated from the ethanol extract of Chamerion angustifolium with normal phase silica column chromatography, Sephadex LH-20, MCI CHP-20 and semi- preparative HPLC methods. Its structure and stereochemistry were elucidated by spectroscopic methods, including 2D-NMR and optical rotation techniques.

[Molecular cloning and characterization of the 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase gene from Artemisia annua L.].

The plastidial methylerythritol phosphate(MEP) pathway provides 5-carbon precursors to the biosynthesis of isoprenoid (including artemisinin). 2-C-Methyl-D-erythritol-4-phosphate cytidylyltransferase (MCT) is the third enzyme of the MEP pathway, which catalyzes 2-C-methyl-D-erythritol-4-phosphate to form 4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol. The full-length MCT cDNA sequence (AaMCT) was cloned and characterized for the first time from Artemisia annua L. Analysis of tissue expression pattern revealed that AaMCT was highly expressed in glandular secretory trichome and poorly expressed in leaf, flower, root and stem. AaMCT was found to be a methyl jasmonate (Me JA)-induced genes, the expression of AaMCT was significantly increased after MeJA treatment. Subcellular localization indicated that the GFP protein fused with AaMCT was targeted specifically in chloroplasts. The transgenic plants of Arabidopsis thaliana with AaMCT overexpression exhibited a significantly increase in the content of chlorophyll a, chlorophyll b and carotenoids, demonstrating that AaMCT kinase plays an influential role in isoprenoid biosynthesis.

[Overexpression of NtPMT and HnH6H changed hyoscyamine-rich Atropa belladonna to scopolamine-rich varieties].

Atropa belladonna L. is the commercial plant material for production of tropane alkaloids, including hyoscyamine and scopolamine. The wild-type Atropa belladonna is characterized by the hyoscyamine-rich chemotype, in which the hyoscyamine content is much higher than the scopolamine content. It is the common goal for the pharmaceutical industry to increase the content of scopolamine in A. belladonna. Based on the T0 progeny of transgenic A. belladonna with NtPMT and HnH6H overexpression, T1 progeny of transgenic A. belladonna were obtained through self-pollination and used in a field trial. The 461 bp fragment of NtPMT and the 1 077 bpHnH6 H were simultaneously expressed from T1 progeny of transgenic A. belladonna, but were not obtained from the wild-type A. belladonna. At the transcription level, the expression of NtPMT and HnH6H were detected in T1 progeny of transgenic A. belladonna, but were not detected in the wild-type plants. Further, the alkaloids were analyzed by HPLC. In the stems and leaves of T1 progeny of transgenic A. belladonna, hyoscyamine was not detected and scopolamine was detected at very high levels; in the stems and leaves of wild-type A. belladonna, hyoscyamine was detected at much higher levels. In the leaves of T1 progeny of transgenic A. belladonna, the content of scopolamine was 15-36 folds higher than that of wild- type leaves; in the stems of T1 progeny of transgenic A. belladonna, the scopolamine content was 37-108 folds higher than that of wild-type stems. In conclusion, overexpression of NtPMT and HnH6H greatly enhanced conversion of hyoscyamine into high-value scopolamine and improved the commercial value of A. belladonna.

[Herpetolide C:one new 7H-dibenzo[c,e]oxepin-5-one from Herpetospermum caudigerum].

The chemical constituents of Herpetospermum caudigerum were investigated using chromatographic methods, including silica gel column chromatography, Sephadex LH-20 and semi-preparative HPLC. Four compounds were isolated and their structures were elucidated by spectral data and physicochemical properties, which were identified as 2,11-dimethoxy-3,9-dihydroxy-7 H-dibenzo[c,e]oxepin-5-one (1),7,8'-didehydroherpetotriol(2), herpetotrio l(3) and kaempferitrin (4). Among those, compound 1 is one new 7H-dibenzo[c,e]oxepin-5-one, named as herpetolide C.

[Molecular cloning and functional characterization of the gene encoding hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase gene from Artemisia annua L.].

Artemisinin is the first choice for malaria treatment. The plastidial MEP pathway provides 5-carbon precursors (IPP and its isomer DMAPP) for the biosynthesis of isoprenoid (including artemisinin). Hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase (HDR) is the last enzyme involved in the MEP pathway, which catalyzes HMBPP to form IPP and DMAPP. In this study, we isolated the full-length cDNA of HDR from Artemisia annua L. (AaHDR2) and performed functional analysis. According to gene expression analysis of AaHDR2 (GenBank: KX058541) and AaHDR1 reported ever (GenBank: ADC84348.1) by qPCR, we found that AaHDR1 and AaHDR2 had much higher expression level in trichomes than that in roots, stems, leaves and flowers. AaHDR2 had much higher expression level in flowers than that in leaves. Further, the plant hormones such as Me JA and ABA respectively up-regulated the expression level of AaHDR1 and AaHDR2 significantly, but GA3 up-regulated the expression level of AaHDR2 only. The gene expression analysis of AaHDR1 and AaHDR2 showed that AaHDR2 had a greater contribution than AaHDR1 to isoprenoid biosynthesis(including artemisinin). We used AaHDR2 for the following experiments. Bioinformatic analysis indicated that AaHDR2 belonged to the HDR family and the functional complementation assay showed that AaHDR2 did have the enzymatic function of HDR, using E. coli mutant MG1655(ara)<>HDR as host cell. The subcellular localization assay showed that AaHDR2 fused with GFP at its N-terminal specifically targeted in chloroplasts. Finally, AaHDR2 was overexpressed in Arabidopsis thaliana. The AaHDR2-overexpressing plants produced the isoprenoids including chlorophyll a, chlorophyll b and carotenoids at significantly higher levels than the wild-type Arabidopsis plants. In summary, AaHDR2 might be a candidate gene for genetic improvement of the isoprenoid biosynthesis.

[Cloning and functional characterization of a cDNA encoding isopentenyl diphosphate isomerase involved in taxol biosynthesis in Taxus media].

Taxol is one of the most potent anti-cancer agents, which is extracted from the plants of Taxus species. Isopentenyl diphosphate isomerase (IPI) catalyzes the reversible transformation between IPP and DMAPP, both of which are the general 5-carbon precursors for taxol biosynthesis. In the present study, a new gene encoding IPI was cloned from Taxus media (namely TmIPI with the GenBank Accession Number KP970677) for the first time. The full-length cDNA of TmIPI was 1 232 bps encoding a polypeptide with 233 amino acids, in which the conserved domain Nudix was found. Bioinformatic analysis indicated that the sequence of TmIPI was highly similar to those of other plant IPI proteins, and the phylogenetic analysis showed that there were two clades of plant IPI proteins, including IPIs of angiosperm plants and IPIs of gymnosperm plants. TmIPI belonged to the clade of gymnosperm plant IPIs, and this was consistent with the fact that Taxus media is a plant species of gymnosperm. Southern blotting analysis demonstrated that there was a gene family of IPI in Taxus media. Finally, functional verification was applied to identify the function of TmIPI. The results showed that biosynthesis of ß-carotenoid was enhanced by overexpressing TmIPI in the engineered E. coli strain, and this suggested that TmIPI might be a key gene involved in isoprenoid/terpenoid biosynthesis.

[Study on medicinal plant resources and diversity in Rhinopithecus bieti national natural reserve of Markam in Tibet].

This research was a part of the investigation of traditional Chinese medicine resources survey in Markam. The medicinal plants in natural reserve were studied for the first in this paper. There were 300 species in 202 genera of 54 families, among them there were 7 species of ferns in 5 genera of 5 families, 6 species of gymnosperms in 4 genera of 3 families, and 287 species of angiosperms in 194 genera of 61 families. There were 166 species Tibetan medicinal plants in 102 genera of 47 families. Quantitative analysis was carried out in 6 aspects of family and genus composition, medicinal parts, drug properties, flavour of a drug, Tibetan medicine, toxicity and new plants. The concrete suggestions of protection and exploitation were put forward, which provided scientific basis for the sustainable utilization of medicinal plants in this area.

[Analysis of photosynthetic characteristics and its influencing factors of medicinal plant Mirabilis himalaica].

To study photosynthetic characteristics and its influencing factors in leaves of medicinal plant Mirabilis himalaica, and provide an evidence for guiding artificial planting and improving the quantity. The light-response and diurnal photosynthesis course of leaves at the booting stages of 1-3 year old M. himalaica were measured with LI-6400 system. The Results showed that the light response curves were fitted well by non rectangle hyperbola equation (R2 > or = 0.98). The values of the maximum photosynthetic rate (Pmax) and light use efficiency of three-year old M. himalaica leaves were higher than those of 1-2 year old individuals. The diurnal variation of net photosynthetic rate (Pn) and stomatal conductance (Gs) of 2-3 year old M. himalaica were typical double-peak curves determinately regulated by stomatal conductance. However, transpiration rate (Tr) of 1-3 year old plants leaves were single-peak curve, which was self-protection of harm reduction caused by the higher temperature at noontime. Correlation analysis showed that the changes between photosynthetic active radiation (PFD), air temperature (T ) and Pn, were significant positive related. Therefore, M. himalaica is a typical sun plant, which should be planted under the sufficient sunshine field and prolong the growing ages suitably in order to improve the yield.

Dracotangusions A and B, two new sesquiterpenes from Dracocephalum tanguticum Maxim. with anti-inflammatory activity.

Two new guaiane sesquiterpenoids were isolated from the dried aerial parts of Dracocephalum tanguticum Maxim., named as dracotangusions A (1) and B (2), together with four known sesquiterpenoids, which were identified as Curcumenone (3), (4Z,7Z,9Z)-11-Hydroxy-4,7,9-germacratriene-1,6-dione (4), Kobusone (5), and (1S,10S), (4S, 5S)-(+)-germacrone-1(10)-4-diepoxide (6). The structures of isolates were determined by UV, IR, HR-ESI-MS, and NMR analysis. What is noteworthy is that four known sesquiterpenoids were isolated for the first time from the genus of Dracocephalum L. All compounds inhibited the extremely significant difference (p < 0.01) in anti-inflammatory activity, suggesting that these compounds may be promising candidates as an anti-inflammatory agent.

Salidroside protects pulmonary artery endothelial cells against hypoxia-induced apoptosis via the AhR/NF-κB and Nrf2/HO-1 pathways.

BACKGROUND: The apoptosis of pulmonary artery endothelial cells (PAECs) is an important factor contributing to the development of pulmonary hypertension (PH), a serious cardio-pulmonary vascular disorder. Salidroside (SAL) is a bioactive compound derived from an herb Rhodiola, but the potential protective effects of SAL on PAECs and the underlying mechanisms remain elusive. PURPOSE: The objective of this study was to determine the role of SAL in the hypoxia-induced apoptosis of PAECs and to dissect the underlying mechanisms. STUDY DESIGN: Male Sprague-Dawley (SD) rats were subjected to hypoxia (10% O2) for 4 weeks to establish a model of PH. Rats were intraperitoneally injected daily with SAL (2, 8, and 32 mg/kg/d) or vehicle. To define the molecular mechanisms of SAL in PAECs, an in vitro model of hypoxic cell injury was also generated by exposed PAECs to 1% O2 for 48 h. METHODS: Various techniques including hematoxylin and eosin (HE) staining, immunofluorescence, flow cytometry, CCK-8, Western blot, qPCR, molecular docking, and surface plasmon resonance (SPR) were used to determine the role of SAL in rats and in PAECs in vitro. RESULTS: Hypoxia stimulation increases AhR nuclear translocation and activates the NF-κB signaling pathway, as evidenced by upregulated expression of CYP1A1, CYP1B1, IL-1ß, and IL-6, resulting in oxidative stress and inflammatory response and ultimately apoptosis of PAECs. SAL inhibited the activation of AhR and NF-κB, while promoted the nuclear translocation of Nrf2 and increased the expression of its downstream antioxidant proteins HO-1 and NQO1 in PAECs, ameliorating the hypoxia-induced oxidative stress in PAECs. Furthermore, SAL lowered right ventricular systolic pressure, and decreased pulmonary vascular remodeling and right ventricular hypertrophy in hypoxia-exposed rats. CONCLUSIONS: SAL may attenuate the apoptosis of PAECs by suppressing NF-κB and activating Nrf2/HO-1 pathways, thereby delaying the progressive pathology of PH.

[Enhancement of tropane alkaloids production in transgenic hair roots of Atropa belladonna by overexpressing endogenous genes AbPMT and AbH6H].

Atropa belladonna L. is the officially medicinal plant species and the main commercial source of scopolamine and hyoscyamine in China. In this study, we reported the simultaneous overexpression of two functional genes involved in biosynthesis of scopolamine, which respectively encoded the upstream key enzyme putrescine N-methyltransferase (PMT; EC 2.1.1.53) and the downstream key enzyme hyoscyamine 6beta-hydroxylase (H6H; EC 1.14.11.11) in transgenic hair root cultures of Atropa belladonna L. HPLC results suggested that four transgenic hair root lines produced higher content of scopolamine at different levels compared with nontransgenic hair root cultures. And scopolamine content increased to 8.2 fold in transgenic line PH2 compared with that of control line; and the other four transgenic lines showed an increase of scopolamine compared with the control. Two of the transgenic hair root lines produced higher levels of tropane alkaloids, and the content increased to 2.7 fold in transgenic line PH2 compared with the control. The gene expression profile indicated that both PMT and H6H expressed at a different levels in different transgenic hair root lines, which would be helpful for biosynthesis of scopolamine. Our studies suggested that overexpression of A. belladonna endogenous genes PMT and H6H could enhance tropane alkaloid biosynthesis.

[An investigation on rare and endangered Tibetan medicinal plants in Lhasa region].

OBJECTIVE: To investigate and study the endangered Tibetan medicinal plant species, their moisture content, biomass and resources reserves in Lhasa region. METHOD: The rare and endangered Tibetan medicinal plant resources were investigated by plot-quadrat method, walking and inquiry ways, sampling and drying method. RESULT: There were 37 species of rare and endangered plants, belonging to 22 families and 34 genera in Lhasa region. The moisture content of aerial part was higher than that of underground part in many plants. The moisture content of Przewalskia tangutica was the highest (91.97%), and the lowest one was Fritillaria delavayi (only 25.99%). The mean biomass of Rubus biflorus was the highest (1 830.480 g), that of Cordyceps sinensis was the lowest (0.291 g). The root-shoot ratio of Asparagus filicinus was the maximum (5.313), the minimum was Aconitum gymnandrum (0.286). The largest output was 18.000 kg x hm(-2) for Berberis agricola, the output of Saxifraga pasumensis was the lowest (0.007 kg x hm(-2)). The resources reserves of the rare and endangered plants were 15683.697 t in Lhasa region, the maximum was 7690.230 t for B. agricola, 49.03% of the total reserves, the minimum was 2.393 t for S. pasumensis, only 0.015%. CONCLUSION: The characteristics of rare and endangered plants were as follows: abundant species and complex habitats, widely distribution but uneven, rich reserves and high economic value. We suggested to update the endangered level of medicinal plants, strengthen the scientific research on these plants, maintain sustainable utilization of the rare and endangered plants in Lhasa region.

[Enhanced biosynthesis of scopolamine in transgenic Atropa belladonna by overexpression of h6h gene].

Transgenic Atropa belladonna with high levels of scopolamine was developed by metabolic engineering. A functional gene involved in the rate limiting enzyme of h6h involved in the biosynthetic pathway of scopolamine was over expressed in A. belladonna via Agrobacterium-mediation. The transgenic plants were culturing till fruiting through micropropogating and acclimating. The integration of the h6h genes into the genomic DNA of transgenic plants were confirmed by genomic polymerase chain reaction (PCR) analysis. Analysis of the difference of plant height, crown width, stem diameter, leaf length, leaf width, branch number and fresh weight was carried out using SPSS software. The content of hyoscyamine and scopolamine in roots, stems, leaves and fruits was determined by HPLC. The investigation of the expression levels of Hnh6h by qPCR. Both Kan(r) and Hnh6h genes were detected in five transgenic lines of A. belladonna plants (A8, A11, A12, C8 and C19), but were not detected in the controls. The plant height, crown width, stem diameter, leaf length, leaf width, branch number and fresh weight of transgenic plants did not decrease by comparison with the non-transgenic ones, and furthermore some agronomic characters of transgenic plants were better than those of the controls. The highest level of scopolamine was found in leaves of transgenic A. belladonna, and the content of scopolamine was also higher than that of hyoscyamine in leaves. The contents of scopolamine of leaves in different transgenic lines were listed in order: C8 > A12 > C19 > A11 > A8, especially, the content of scopolamine in transgenic line C8 was 2.17 mg x g(-1) DW that was 4.2 folds of the non-transgenic ones (0.42 mg x g(-1) DW). The expression of transgenic Hnh6h was detected in all the transgenic plants but not in the control. The highest level of Hnh6h expression was found in transgenic leaves. Overexpression of Hnh6h is able to break the rate limiting steps involved in the downstream pathway of scopolamine biosynthesis, and thus promotes the metabolic flux flowing toward biosynthesis of scopolamine to improve the capacity of scopolamine biosynthesis in transgenic plants. As a result, transgenic plants of A. belladonna with higher level of scopolamine were developed.

Structures and immunomodulatory activity of one galactose- and arabinose-rich polysaccharide from Sambucus adnata.

One galactose- and arabinose-rich polysaccharide isolated from Sambucus adnata was named SPS-1, which had an average molecular weight 138.52 kDa, and was composed of L-rhamnose, D-glucuronic acid, D-galacturonic acid, D-galactose, and L-arabinose in a molar ratio of 0.6:0.4:0.1:4.9:4.0. The primary structure of SPS-1 was further analyzed through methylation and NMR spectroscopy. The results showed that SPS-1 had the structural characteristics of AG-II pectin. The immunoactivity test showed that SPS-1 activated the phosphorylation of MAPKs-related proteins and further elevated the expression levels of related nuclear transcription factors (IκBα and NF-κB p65) in the cells through the TLR2 and MyD88/TRAF6-dependent pathway, thereby significantly enhancing the phagocytosis of macrophages and stimulating the secretion of NO, IL-1ß, IL-6, and TNF-α, which activated the RAW264.7 cells. Therefore, SPS-1, acting as an immunomodulator, is a potential drug for immunological diseases.