Identification of Flavonoids from Scutellaria barbata D. Don as Inhibitors of HIV-1 and Cathepsin L Proteases and Their Structure-Activity Relationships.

Scutellaria barbata D. Don (SB, Chinese: Ban Zhi Lian), a well-known medicinal plant used in traditional Chinese medicine, is rich in flavonoids. It possesses antitumor, anti-inflammatory, and antiviral activities. In this study, we evaluated the inhibitory activities of SB extracts and its active components against HIV-1 protease (HIV-1 PR) and SARS-CoV2 viral cathepsin L protease (Cat L PR). UPLC/HRMS was used to identify and quantify the major active flavonoids in different SB extracts, and fluorescence resonance energy transfer (FRET) assays were used to determine HIV-1 PR and Cat L PR inhibitions and identify structure-activity relationships. Molecular docking was also performed, to explore the diversification in bonding patterns of the active flavonoids upon binding to the two PRs. Three SB extracts (SBW, SB30, and SB60) and nine flavonoids inhibited HIV-1 PR with an IC50 range from 0.006 to 0.83 mg/mL. Six of the flavonoids showed 10~37.6% inhibition of Cat L PR at a concentration of 0.1 mg/mL. The results showed that the introduction of the 4'-hydroxyl and 6-hydroxyl/methoxy groups was essential in the 5,6,7-trihydroxyl and 5,7,4'-trihydroxyl flavones, respectively, to enhance their dual anti-PR activities. Hence, the 5,6,7,4'-tetrahydroxyl flavone scutellarein (HIV-1 PR, IC50 = 0.068 mg/mL; Cat L PR, IC50 = 0.43 mg/mL) may serve as a lead compound to develop more effective dual protease inhibitors. The 5,7,3',4'-tetrahydroxyl flavone luteolin also showed a potent and selective inhibition of HIV-1 PR (IC50 = 0.039 mg/mL).

[Identification of the Strain Which Highly Produces Protease and ß-D-glucosidase Isolated from Shuidouchi Produced in Sichuan and Evaluating Its Ability of Producing Protease].

OBJECTIVE: To select and identify the bacterium which highly produces protease and ß-D-glucosidase from 72 strains of Shuidouchi from Sichuan, and to provide evidence for further research on its nutritional value and fermentation strain exploiting. METHODS: Casein degradation test and pNPG chemical test were applied respectively to detect the capacity to produce protease and ß-D-glucosidase of each strain. Characteristics of morphology, biochemistry, 16S rRNA and MALDI-TOF-MS were used to identify the fermentation strain, which genetic stability, curves of growth and enzyme producing were also obtained. RESULTS: The strain with the highest enzyme activity of ß-D-glucosidase (0.084 U/L) among the top 10 strains for producing protease was selected as the fermentation strain and was identified as Bacillus subtilis, which curves of growth and enzyme producing conformed as well. The result of genetic stability showed that capacity of enzyme producing was stable until the 10th generation. CONCLUSIONS: The fermentation strain which highly produced protease and ß-D-glucosidase was selected from 72 strains of shuidouchi from Sichuan and was identified as Bacillus subtilis.

[Genome survey and characteristic analysis of SSR in Callicarpa nudiflora].

Callicarpa nudiflora,which is a big brand of Li nationality medicine with Hainan characteristics,has the effects of dissolving stasis,hemostasis,anti-inflammatory and antibacterial. At present,there is a lack of information about the reference genome of C. nudiflora. The study of the genome size,heterozygosity rate and characteristics of SSR of C. nudiflora,can provide an effective basis for the formulation of the whole genome de novo sequencing strategy and development of SSR molecular markers of C. nudiflora. To realize this purpose,high throughput sequencing platform Illumina Hiseq was used to sequence the genome structure of C. nudiflora and K-mer analysis was applied to estimate genome size,repeat sequences and heterozygosity rate. Simple-sequence repeat( SSR) loci that are suitable as markers were identified by MISA software. The results showed the estimated genome size of C. nudiflora was 822. 43 Mb,with a 0. 85% heterozygosity rate and 71. 67% repeats,and the GC content of genome was about 49. 20%. Therefore,C. nudiflora belongs to a complex genome with high heterozygosity and repetition. SSR molecular genetic markers were analyzed in the genome sequence,and a total of 206 049 SSRs were identified,among which mono-nucleotide,di-nucleotide and tri-nucleotide repetitive motifs summed up to 198 993,accounting for 96. 57% of the total SSRs. Among the 2-6 nucleotide repeats,AT/AT,AAT/ATT,AGCC/CTGG,AAAAT/ATTTT and AGATAT/ATATCT have the largest number,respectively. This report represents the first genome-wide characterization of C. nudiflora,and provides a reference for the construction of the library for the fine sequencing of the genome,and a molecular basis for the development of SSR molecular markers as well as for the protection and utilization of gene resources.

NIR Rapid Assessments of Blumea balsamifera (Ai-na-xiang) in China.

Blumea balsamifera (Ai-na-xiang) is used as an important plant source of natural borneol, which is widely used in the pharmaceutical industry. The aim of this study was to establish the methods based on near infrared (NIR) spectroscopy for determining the geographical origin of B. balsamifera, as well as developing a method for the quantitative rapid analysis of the active pharmaceutical ingredients (APIs). A total of 109 samples were collected from China in 2013 and arbitrarily divided into calibration and prediction sets using the Kennard-Stone algorithm. The l-borneol and total flavone contents of the samples were measured by gas chromatography and ultraviolet-visible spectroscopy, respectively. The NIR spectra were acquired using an integrating sphere and a partial least squares (PLS) model was built using the optimum wavelength regions, which were selected using a synergy interval partial least-squares (SiPLS) algorithm. The root mean square errors of prediction of the l-borneol and total flavone models were 0.0779 and 2.2694 mg/g, with R² of 0.9069 and 0.8013, respectively. A discriminant model to determine the geographical origin of B. balsamifera (Guizhou and Hainan) was also established using a partial least squares discriminant analysis method with an optimum pretreatment method. The prediction accuracy rate of the model was 100%. NIR spectroscopy can be used as a reliable and environmentally friendly method to determine the API and the origin of different B. balsamifera samples.

Variations in Essential Oil Yield, Composition, and Antioxidant Activity of Different Plant Organs from Blumea balsamifera (L.) DC. at Different Growth Times.

Blumea balsamifera, also named Ainaxiang, is widely used as an ancient medicinal herb in tropical and subtropical Asia. It is rich in essential oils. In this work the essential oils of B. balsamifera from different plant organs and in different months were extracted, and then analyzed by gas chromatography-mass spectrometry. The results showed that essential oil yield of young leaves was the highest (0.65 mL/100 g), followed by mature leaves (0.57 mL/100 g), and the oil yield was higher in October (0.47 mL/100 g) than other months. A total of 44 compounds were identified, representing 92.64%-96.71% of the oil. Eighteen common chemical components were found among the six plant organs, representing >80% of the oil constituents. l-borneol was the main ingredient in leaves, and its content was the highest in senescent leaves and in December. In the essential oils of young shoots and young stems, the main component was dimethoxydurene. Antioxidant activity was also determined using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) and ß-carotene bleaching (BCB) assays. The results indicated that the ß-carotene bleaching activity was far stronger than the DPPH radical-scavenging capacity, and the young leaves and young shoots showed stronger antioxidant activity. Dimethoxydurene, ß-caryophyllene, and α-caryophyllene play a positive role in good antioxidant activity, while ß-eudesmol, phytol, and tetradecanal play a negative role. The antioxidant activity revealed in this study might help in developing this promising bioresource for use in the medicinal and cosmetic industries.

[Metabolic pathway of flavonoids in Blumea balsamifera].

To better understand the biosynthetic pathway of flavonoids in Blumea balsamifera, and to increase the biosynthesis of flavone and flavonol amount, we carried out this research. Basing on transcriptome information, B. balsamifera flavonoids metabolic pathway was analyzed in KEGG data base. The result demonstrated that two metabolic pathways of flavonoids were found in KEGG data base. They were flavonoid biosynthesis pathway (No.ko00941) that contained 32 genes and flavone and flavonol biosynthesis pathway (No.ko00944) that contained 12 genes. Metabolic pathway of flavonoids in B. balsamifera was similar to that in other plants. Chalcone synthase (CHS) and Chalconeisomerase were much likely to be key enzymes on metabolic pathway of flavonoids in B. balsamifera. HCT was much relevant to biosynthesis of favones.

[Analysis of metabolic pathway of terpenoids in Blumea balsamifera].

In order to provide a theoretical basis for the regulation of active ingredient, the terpenoids metabolic pathway and specific enzymes in Blumea balsamifera are investigated. Basing on transcriptome information, B. balsamifera terpenoids metabolic pathway was analyzed in KEGG data base. Four metabolic pathway of terpenoids were found in KEGG data base. They were terpenoid backbone biosynthesis, monoterpenoid biosynthesis, diterpenoid biosynthesis, sesquiterpenoid and triterpenoid biosynthesis, contained 103, 10, 29,59 genes, respectively. Through the analysis of the enzyme and product in the pathway, the result showed that there were 8 kinds of monoterpenes, 3 kinds of diterpenes, 3 kinds of triterpenes and sesquiterpenes. The mainly key enzymes were deoxyxylulose 5-phosphate synthase, HMG-CoA reductase and allyl transferase system. In B. balsamifera, there were relatively few monoterpenes synthetic enzymes, while the type of products was much more than other terpenes. This may be relate to the non-specific catalytic characteristic of monoterpene synthase. It is expected to improve the yield of terpenoids in B. balsamifera by analysis the pathways and regulation the key enzymes.

Blumea balsamifera Oil for the Acceleration of Healing of Burn Injuries.

Blumea balsamifera oil (BBO) is a main extract obtained from Blumea balsamifera (L.) DC (Ainaxiang) leaves, which are widely used as a traditional medicine by the Miao and Li Nations to promote skin trauma or burn injury healing. This study was initiated to investigate the healing efficacy in deep second-degree burn model in rats. The rats were treated by BBO for 21 consecutive days. The rate of healing, scabs dropped time and re-epithelialization time were observed every three days for 21 days after burn injury. The samples were collected from different treated rats by sacrificing the animals on the 1st, 2nd, 5th, 9th, 14th, and 21st day post-burn creation. Then, the water content of burn tissue was measured. Plasma interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF-α) levels were evaluated, and the tissue expressions of basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), and transforming growth factor-beta (TGF-ß) were determined along with skin histopathology. The results showed that the water content of tissue was significantly reduced, the scabs dropped time shortened, and healing accelerated after treatment with BBO in the burn injury rats. Furthermore, the expressions of growth factors were significantly increased in the tissue; however, the levels of inflammatory factors on plasma decreased. This study confirms the efficacy of BBO consumption on burn injuries.

External application of the volatile oil from Blumea balsamifera may be safe for liver--a study on its chemical composition and hepatotoxicity.

Ainaxiang (Blumea balsamifera), also known as Sambong, is an important ancient medicinal herb in Southeast Asia. It is rich in volatile oil, and still widely used nowadays for skin wound healing and treatment of sore throats. We analyzed the volatile oil from Blumea balsamifera (BB oil) by gas chromatography-mass spectrometry (GC-MS). Forty one components, including l-borneol, were identified. Next, the damaging effects of BB oil diluted with olive oil on liver at different concentrations (100%, 50%, 20%), were evaluated, using both normal and wounded skin. Plasma ALT, AST, ALP and TBili were assessed, along with liver histopathology. The results showed that serum levels of liver toxicity markers in the high concentration groups (100% w/v) increased compared with control groups, whereas no significant changes was observed in histopathology of liver samples. In the wound groups, treatment with BB oil resulted in a decrease in serum toxicity index, compared with normal animal groups. This study confirms the safety of short term BB oil consumption, though high BB oil doses may lead to mild liver injury and this response might be weakened in the case of cutaneous wounds. These results are expected to be helpful for guiding appropriate therapeutic use of BB oil.

[Genetic potential and selection index of yield-related traits in 2-year-old licorice (Glycyrrhiza uralensis) populations].

OBJECTIVE: To evaluate genetic potential and selection index of yield-related traits in 2-year-old populations, and provide a basis for high-yield breeding of licorice (Glycyrrhiza uralensis). METHODS: Four genotype licorice populations were transplanting in four different environment using complete randomized block design with three replication, and the 10 yield-related traits, including plant height (PH), stem diameter (SD), tiller number (TN), taproot length (TRL), root length (RL), root diameter (RD), diameter of 20 cm below the root head (D20), taperingness (TR), lateral root number (LRN) and root fresh weight (RFW) were measured in field. Genetic potential and selection index of yield-related traits were evaluated using theories of quantitative genetics. RESULTS: The genetic potential were higher for the three traits of PH (X1), TRL (X4) and RL (X5) that showed through genetic parameter of broad sense heritability (h2), genetic variation coefficient (GCV) and relative genetic advance (RGA). Among them, selection relative value increased by 45.94% through combination of PH and RL (Y1 = 0.21X1 + 0.08X5) compared with the direct selection for RFW (Y1). CONCLUSION: It can realize effective indirectly selection for yield traits, RFW of licorice by PH and RL or their combination. That is, for selection of high-yield licorice, select big and high plants firstly, and then single out long root length materials.

[Cloning and expression regulation of 1-deoxy-D-xylulose-5-phosphate reductoisomerase cDNA from Alpinia officinarum].

The rhizome of Alpinia officinarum is a widely used Chinese herbal medicine. The essential oil in A. officinarum rhizome is mainly composed of 1, 8-cineole and other monoterpenes, as the major bioactive ingredients. In plants, monoterpenes are synthesized through the methylerythritol phosphate (MEP) pathway in the plastids, and 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) is an enzyme catalyzing a committed step of the MEP pathway. In the present study, the full-length cDNA encoding DXR was cloned from the rhizome of A. officinarum, using homology-based RT-PCR and rapid amplification of cDNA ends (RACE) techniques. The new cDNA was designated as AoDXR and submitted to GenBank to be assigned with an accession number HQ874658. The full-length cDNA of AoDXR was 1 670 bp containing a 1 419 bp open reading frame encoding a polypeptide of 472 amino acids with a calculated molecular mass of 51.48 kDa and an isoelectric point of 6.15. Bioinformatic analyses revealed that AoDXR showed extensive homology with DXRs from other plant species and contained a conserved plastids transit peptide, a Pro-rich region and two highly conserved NADPH-binding motifs in its N-terminal region characterized by all plant DXRs. The phylogenetic analysis revealed that AoDXR belonged to angiosperm DXRs. The structural modeling of AoDXR showed that AoDXR had the typical V-shaped structure of DXR proteins. The tissue expression pattern analysis indicated that AoDXR expressed strongly in leaves, weak in rhizomes of A. officinarum. Exogenous methyl jasmonate (MeJA) could enhance the expression of AoDXR and the production of 1, 8-cineole in A. officinarum rhizomes. The cloning and characterization of AoDXR will be helpful to reveal the molecular regulation mechanism of monoterpene biosynthesis in A. officinarum and provides a candidate gene for metabolic engineering in improving the medicinal quality of A. officinarum rhizome.

Engineering PD-L1 Cellular Nanovesicles Encapsulating Epidermal Growth Factor for Deep Second-Degree Scald Treatment.

Scars are common and intractable consequences after scalded wound healing, while monotherapy of epidermal growth factors does not solve this problem. Maintaining the stability of epidermal growth factors and promoting scarless healing of wounds is paramount. In this study, engineering cellular nanovesicles overexpressing PD-L1 proteins, biomimetic nanocarriers with immunosuppressive efficacy, were successfully prepared to encapsulate epidermal growth factors for maintaining its bioactivity. Remarkably, PD-L1 cellular nanovesicles encapsulating epidermal growth factors (EGF@PDL1 NVs) exerted desired therapeutic effect by attenuating the overactivation of T cell immune response and promoting skin cells migration and proliferation. Hence, EGF@PD-L1 NVs promoted wound healing and prevented scarring in deep second-degree scald treatment, demonstrating a better effect than using individual PD-L1 NVs or EGF. This research proved that EGF@PD-L1 NVs is considered an innovative and thorough therapy of deep second-degree scald.

Phylogenetic Reconstruction and Divergence Time Estimation of Blumea DC. (Asteraceae: Inuleae) in China Based on nrDNA ITS and cpDNA trnL-F Sequences.

The genus Blumea is one of the most economically important genera of Inuleae (Asteraceae) in China. It is particularly diverse in South China, where 30 species are found, more than half of which are used as herbal medicines or in the chemical industry. However, little is known regarding the phylogenetic relationships and molecular evolution of this genus in China. We used nuclear ribosomal DNA (nrDNA) internal transcribed spacer (ITS) and chloroplast DNA (cpDNA) trnL-F sequences to reconstruct the phylogenetic relationship and estimate the divergence time of Blumea in China. The results indicated that the genus Blumea is monophyletic and it could be divided into two clades that differ with respect to the habitat, morphology, chromosome type, and chemical composition of their members. The divergence time of Blumea was estimated based on the two root times of Asteraceae. The results indicated that the root age of Asteraceae of 76-66 Ma may maintain relatively accurate divergence time estimation for Blumea, and Blumea might had diverged around 49.00-18.43 Ma. This common ancestor had an explosive expansion during the Oligocene and Miocene and two major clades were differentiated during these epochs 29.60 Ma (17.76-45.23 Ma 95% HPD (Highest Posterior Density). Evidence from paleogeography and paleoclimate studies has confirmed that Blumea experienced differentiation and an explosive expansion during the Oligocene and Miocene.

UPLC-QTOF-MS identification of metabolites in rat biosamples after oral administration of Dioscorea saponins: a comparative study.

ETHNOPHARMACOLOGICAL RELEVANCE: Among the 49 species of the genus Dioscorea distributed in China, Dioscorea nipponica Makino (DN), Dioscorea panthaica Prain et Burkill (DP), and Dioscorea zingiberensis C. H. Wright (DZ) possess more or less similar traditional therapeutic actions, such as activating blood, relieving pain, and dispersing swelling; they have been used as folk medicine in China since 1950s. The modern pharmaceutical industry has developed these three species as herbal medicines that have been used for decades for treating cardiovascular diseases. However, there is no available information in the literature explaining how their chemical components are converted and interrelated in vivo to support their efficacies. The present study aimed to a) compare the metabolic profiles of saponins from DN, DP and DZ, which are considered to be their bioactive components, and b) to compare the changes in sustained levels of metabolites from rat biosamples. MATERIAL AND METHODS: Total saponins (TS) from each of the three species, and four individual saponins, namely protodioscin (PD), pseudoprotodioscin (PSD), dioscin (DC) and diosgenin (DG), were given to rats by oral administration. Chemical profiles of the rats' plasma, urine and feces were monitored 1-36 h. A UPLC-QTOF-MS based method was performed to identify the absorbed constituents and their metabolic products in rat biosamples (i.e., blood, urine, and feces); the ratio of peak area of major saponins to that of internal standard was calculated and plotted versus time to characterize the sustained levels of saponins in biosamples. RESULTS: Totally 10 saponin-related compounds were detected in rat plasma, 10 in rat urine and 18 in rat feces. The results indicated that formation of diosgenin by desugarization was the main pathway by which steroidal glycosides were metabolized. Other types of bio-transformation were found among glycosides and aglycones, such as ring cyclization through loss of 26-O-glucosyl, substitution of ß-D-glucopyranosyl for α-L-rhamnopyrannosyl, hydrogenation of diosgenin at 5(6)-double bond, and hydration of 20(22)-double bond. Generally, the metabolic profiles of DN and DP were shown to be quite similar, but different from that of DZ. However, some particular similarities and connections were found among these three TS. Diosgenin was one of the main metabolites commonly found in plasma and feces (excluding urine), from all groups receiving different TS, as well as individual saponins; this is likely to be one of the bioactive constituents playing an essential role in cardioprotective efficacy. Furostane-type saponins in TS of DN, DP or DZ, such as PD, protogracillin, parvifloside, protodeltonin and protobioside, showed fast absorption into blood (<1h), but were maintained for a relatively short period (mostly<8h), while the spirostane-type saponin and sapogenin (DC and DG, respectively), were absorbed into circulation more slowly (>1h), but increased gradually and lasted longer (>36h). These two patterns suggest that the therapeutic effect of these Dioscorea saponins is achieved through a complex, multi-step process over time. In addition, it appears that PD, PSD, and DC contained in DN and DP were transformed into certain glycosides originally found in DZ but not in DN or DP (protodeltonin, deltonin, trillin, and progenin II), which might indicate another linkage among these three species. CONCLUSION: These similarities and connections described above constitute evidence supporting similarity in efficacy of these three herbs from the perspective of metabolism. The UPLC-QTOF-MS based method is accurate and efficient for analyzing metabolic changes in rat biosamples over time.