Ximin Li

Characterisation of polyphenol constituents of Linderae aggregate leaves using HPLC fingerprint analysis and their antioxidant activities.

The leaves of Linderae aggregate (LAL) has been used as a type of tea in China and other Southeast Asian countries. In this study, 11 polyphenols in LAL were clarified for the first time using multiple high-performance liquid chromatographic techniques. An optimal extracting method was developed through the comparison of the amount of quercetin-3-O-α-L-rhamnoside using a uniform design method. From the fingerprint liquid chromatography data, 11 common peaks in the 8 samples collected from April to November were semi-determined. The antioxidant capacities were examined using the 2,2-diphenyl-1-picrylhydrazyl free radical scavenging assay and the ferric reducing/antioxidant power assay. All 8 samples contained the same 11 polyphenols in similar ratios. Three samples, S2, S5 and S6 contained higher amount of quercetin-3-O-α-L-rhamnoside and were demonstrated to have stronger antioxidant capacities in both antioxidant assays. These results are critical in optimising harvest time and quality control of LAL.

Proteomics analysis of Mahonia bealei leaves with induction of alkaloids via combinatorial peptide ligand libraries

UNLABELLED Alkaloids are one of the most attractive sources for obtaining active natural products. However, alkaloids exist in the plants as the secondary metabolites with tracing amount, and there is an enormous demand for a large production. In the present study, we aimed to profile the modification of benzylisoquinoline alkaloids in Mahonia bealei seedlings under the binary stress of ultraviolet-B irradiation and dark incubation. Comparative proteomics analysis was carried out to address the underlying proteome variations that accounted for the alkaloid induction under treatment. Thirteen differential proteins were identified in the leaves under binary stress. Of note, the abundance of S-adenosyl-L-methionine synthetase was highly increased to sustain a high concentration of S-adenosyl-L-methionine for the enhanced biosynthesis of alkaloids. Additionally, we presented the application of CPLL to M. bealei leaf proteins. Three new secondary metabolism proteins and 12 additional differential proteins were identified only after CPLL treatment. Six genes in the benzylisoquinoline alkaloid biosynthesis pathway were selected to verify their variable expression using quantitative real time polymerase chain reaction. The results suggest that the benzylisoquinoline alkaloids in M. bealei leaf were increased to eliminate the adverse effect of UV-B exposure. The suppression of photosynthesis and respiratory rate may save an extra energy for the secondary metabolites, and the enhanced N-metabolism may supply sufficient primary metabolite precursors. To our best knowledge, this is the first work aimed at the secondary metabolism proteomic characterization of M. bealei using the CPLL technique. It also presented an effective and innovative process to improve the contents of alkaloids in medicinal plants for industrial production. BIOLOGICAL SIGNIFICANCE Besides the effective and innovative process to improve the contents of alkaloids in M. bealei leaves for industrial production, the presented combinatorial hexapeptide ligand library technology was applied for the study of low-abundance protein in medicinal plant. It may be an available tool for the analysis of the secondary proteins.

Binary stress induces an increase in indole alkaloid biosynthesis in Catharanthus roseus

Catharanthus roseus is an important medicinal plant, which produces a variety of indole alkaloids of significant pharmaceutical relevance. In the present study, we aimed to investigate the potential stress-induced increase of indole alkaloid biosynthesis in C. roseus using proteomic technique. The contents of the detectable alkaloids ajmalicine, vindoline, catharanthine, and strictosidine in C. roseus were significantly increased under binary stress. Proteomic analysis revealed that the abundance of proteins related to tricarboxylic acid cycle and cell wall was largely increased; while, that of proteins related to tetrapyrrole synthesis and photosynthesis was decreased. Of note, 10-hydroxygeraniol oxidoreductase, which is involved in the biosynthesis of indole alkaloid was two-fold more abundant in treated group compared to the control. In addition, mRNA expression levels of genes involved in the indole alkaloid biosynthetic pathway indicated an up-regulation in their transcription in C. roseus under UV-B irradiation. These results suggest that binary stress might negatively affect the process of photosynthesis in C. roseus. In addition, the induction of alkaloid biosynthesis appears to be responsive to binary stress.

Proteomics analysis of UV-irradiated Lonicera japonica Thunb. with bioactive metabolites enhancement.

A previous study showed that the contents of caffeoylquinic acids and iridoids, the major bioactive components in the postharvest Lonicera japonica Thunb., were induced by enhanced ultraviolet (UV)‐A or UV‐B irradiation. To clarify the UV‐responsive key enzymes in the bioactive metabolites biosynthetic pathway and the related plant defense mechanism in L. japonica, 2DE in combination with MALDI‐TOF/TOF MS was employed. Seventy‐five out of 196 differential proteins were positively identified. Based on the functions, these proteins were grouped into nine categories, covering a wide range of molecular processes including the secondary metabolites (caffeoylquinic acids and iridoids) biosynthetic‐related proteins, photosynthesis, carbohydrate and energy metabolism, stress, DNA, transport‐related proteins, lipid metabolism, amino acid metabolism, cell wall. Of note is the increasing expression of 1‐deoxy‐d‐xylulose 5‐phosphate reductoisomerase and 5‐enol‐pyruvylshikimate‐phosphate synthase, which was crucial to supply more precursor for the secondary metabolites including caffeoylquinic acids and iridoids. Thus, this study provides both the clues at the protein level for the increase of the two bioactive components upon UV irradiation and the profile of UV‐responsive proteins in L. japonica.

Identification of candidate synovial membrane biomarkers after Achyranthes aspera treatment for rheumatoid arthritis

Rheumatoid arthritis (RA) is a systemic autoimmune disease whose main symptom is a heightened inflammatory response in synovial tissues. To verify the anti-arthritic activities of Achyranthes aspera and its possible therapy-related factors on the pathogenesis of RA, the saponins in A. aspera root were isolated and identified to treat the collagen-induced arthritis (CIA) rats. Phytochemical analysis isolated and identified methyl caffeate, 25-S-inokosterone, 25-S-inokosterone β-D-glucopyranosyl 3-(O-β-D-glucopyranosyloxy)-oleanolate, and β-D-glucopyranosyl 3-(O-β-D-galactopyranosyl (1→2)(O-β-D-glucopyranosyloxy)-oleanolate as main compounds in the root of A. aspera. Proteomics was performed to determine the differentially expressed proteins in either inflamed or drug-treated synovium of CIA rats. Treatment resulted in dramatically decreased paw swelling, proliferation of inflammatory cells, and bone degradation. Fibrinogen, procollagen, protein disulfide-isomerase A3, and apolipoprotein A-I were all increased in inflamed synovial tissues and were found to decrease when administered drug therapy. Furthermore, Alpha-1-antiproteinase and manganese superoxide dismutase were both increased in drug-treated synovial tissues. The inhibition of RA progression shows that A. aspera is a promising candidate for future treatment of human arthritis. Importantly, the total saponins found within A. aspera are the active component. Finally, autoantigens such as fibrinogen and collagen could act as inducers of RA due to their aggravation of inflammation. Given this, it is possible that the vimentin and PDIA3 could be the candidate biomarkers specific to Achyranthes saponin therapy for rheumatoid arthritis in synovial membrane.

Comparative proteomic analysis of the sun- and freeze-dried earthworm Eisenia fetida with differentially thrombolytic activities

UNLABELLED The dried earthworm is a traditional thrombolytic medicine in East Asia. Its thrombolytic mechanism has been extensively studied. However, the effects of drying process on thrombolysis were rarely investigated. Herein, we compared the thrombolytic activity of earthworm Eisenia fetida processed by sun-drying to that by freeze-drying. Fibrin plate and blood clot lysis assays showed that freeze-dried earthworms gave dramatically higher fibrinolytic and thrombolytic activities than the sun-dried earthworms. To address the thrombolytic difference, comparative proteomic analysis was carried out using fibrin zymography and two-dimensional gel electrophoresis (2-DE). The freeze- and sun-dried earthworms generated remarkably different 2-DE protein spot patterns. A total of 126 differential protein spots were detected, 83 of them were identified by matrix-assisted laser desorption/ionization-tandem time-of-flight mass spectrometry and database searching with 13 quantitative changes and 70 qualitative changes. Five of these differential proteins were identified as fibrinolytic proteases (lumbrokinases), responsible for dissolving fibrin, the main protein component of thrombus. The total abundance of these fibrinolytic proteases in the freeze-dried earthworms was significantly higher, consistent with the results of fibrin zymography. Therefore, the higher concentration of fibrinolytic enzymes along with their broad substrate specificity explained the stronger fibrinolytic and thrombolytic activities of the freeze-dried earthworms. This study suggests that freeze-drying represents an improved processing method for earthworm as the thrombolytic therapy in the future. BIOLOGICAL SIGNIFICANCE Thrombosis has become one of the biggest concerns all over the world. The dried earthworms have been intensively used as thrombolytic agents. Its thrombotic mechanism has been studied by the modern pharmacological researches. However, the drying procedure of the earthworm and its effects on the thrombolysis were rarely investigated. The present study compared the thrombolytic effects of the freeze-dried and the normal dried earthworm E. fetida. To better understand the underlying mechanisms for differential thrombolytic effects, the fibrin zymography and the two-dimensional gel electrophoresis (2-DE) were employed to identify sets of differential proteins. Therefore, this study provides not only the comparative proteomic analysis but also molecular mechanism underlying the differential thrombolytic effects.

Response and Defense Mechanisms of Taxus chinensis Leaves Under UV-A Radiation are Revealed Using Comparative Proteomics and Metabolomics Analyses

Taxus chinensis var. mairei is a species endemic to south-eastern China and one of the natural sources for the anticancer medicine paclitaxel. To investigate the molecular response and defense mechanisms of T. chinensis leaves to enhanced ultraviolet-A (UV-A) radiation, gel-free/label-free and gel-based proteomics and gas chromatography-mass spectrometry (GC-MS) analyses were performed. The transmission electron microscopy results indicated damage to the chloroplast under UV-A radiation. Proteomics analyses in leaves and chloroplasts showed that photosynthesis-, glycolysis-, secondary metabolism-, stress-, and protein synthesis-, degradation- and activation-related systems were mainly changed under UV-A radiation. Forty-seven PSII proteins and six PSI proteins were identified as being changed in leaves and chloroplasts under UV-A treatment. This indicated that PSII was more sensitive to UV-A than PSI as the target of UV-A light. Enhanced glycolysis, with four glycolysis-related key enzymes increased, provided precursors for secondary metabolism. The 1-deoxy-d-xylulose-5-phosphate reductoisomerase and 4-hydroxy-3-methylbut-2-enyl diphosphate reductase were identified as being significantly increased during UV-A radiation, which resulted in paclitaxel enhancement. Additionally, mRNA expression levels of genes involved in the paclitaxel biosynthetic pathway indicated a down-regulation under UV-A irradiation and up-regulation in dark incubation. These results reveal that a short-term high dose of UV-A radiation could stimulate the plant stress defense system and paclitaxel production.

Improved metabolites of pharmaceutical ingredient grade Ginkgo biloba and the correlated proteomics analysis

Ginkgo biloba is an attractive and traditional medicinal plant, and has been widely used as a phytomedicine in the prevention and treatment of cardiovascular and cerebrovascular diseases. Flavonoids and terpene lactones are the major bioactive components of Ginkgo, whereas the ginkgolic acids (GAs) with strong allergenic properties are strictly controlled. In this study, we tested the content of flavonoids and GAs under ultraviolet‐B (UV‐B) treatment and performed comparative proteomic analyses to determine the differential proteins that occur upon UV‐B radiation. That might play a crucial role in producing flavonoids and GAs. Our phytochemical analyses demonstrated that UV‐B irradiation significantly increased the content of active flavonoids, and decreased the content of toxic GAs. We conducted comparative proteomic analysis of both whole leaf and chloroplasts proteins. In total, 27 differential proteins in the whole leaf and 43 differential proteins in the chloroplast were positively identified and functionally annotated. The proteomic data suggested that enhanced UV‐B radiation exposure activated antioxidants and stress‐responsive proteins as well as reduced the rate of photosynthesis. We demonstrate that UV‐B irradiation pharmaceutically improved the metabolic ingredients of Ginkgo, particularly in terms of reducing GAs. With high UV absorption properties, and antioxidant activities, the flavonoids were likely highly induced as protective molecules following UV‐B irradiation.

The complete chloroplast genome sequence of Hydrocotyle sibthorpioides (Apiales: araliaceae)

Abstract Hydrocotyle sibthorpioides Lam. is a widespread and important Chinese medicinal plant. In this study, we have sequenced the complete chloroplast genome of H. sibthorpioides, which is 152,880 bp in length with large (LSC 84,064 bp) and small (SSC 18,690 bp) single-copy regions separated by a pair of inverted repeats (IRs 25,063 bp) and contains 113 unique genes with 17 genes duplicated in the IR making a total of 130 genes. The phylogenetic analysis strongly supports the position of H. sibthorpioides and indicates it belongs to the Araliaceae family, potential to facilitate a better understanding of population and phylogenetic studies in Apiales.

Phytochemical compositions, antioxidant and antimicrobial activities analysis of extracts from Vaccinium bracteatum Thunb. leaves

Vaccinium bracteatum Thunb. is an edible plant, which has been used for many food products and is also a resource of traditional Chinese medicine. In this study, the antioxidant and antimicrobial activities of ethanol extracts from its leaves were investigated. To study the characteristic compositions, twelve compounds of extracts accumulated by the D-101 macroporous adsorption resin (VBE) were identified by HPLC-DAD and HPLC-ESI/MS techniques, including chlorogenic acid and its isomers, and eight flavonoid compounds. The contents of total flavonoids, orientin and isoorientin in the accumulated part were 601.4, 44.7, and 96.1 mg/g, respectively, which were far more than that in the raw materials. Furthermore, the antioxidant activities were estimated by DPPH, ABTS, and FRAP assays, which showed that the high content accompanied with strong antioxidant activities. Besides, compared to the same type of bamboo leaves (AOB), the accumulated part possesses better activities. At the last, the antimicrobial activities of VBE were assessed by a serial two-fold dilution assay, the results showed that it had good antimicrobial activities. Taken together, extracts from Vaccinium bracteaturn Thunb. leaves have better antioxidant activities, which can be used as a natural antioxidant.