Biosynthesis of Silver Nanoparticles from Duchesnea indica Extracts Using Different Solvents and Their Antibacterial Activity.

Silver nanoparticles (AgNPs) were synthesized using the whole plant of Duchesnea indica (DI) which was extracted in different solvents; the antimicrobial effects of the extract were investigated in this study. The extraction of DI was performed using three different solvents: water, pure ethanol (EtOH), and pure dimethyl sulfoxide (DMSO). AgNP formation was monitored by measuring the UV-Vis spectrum of each reaction solution. After synthesis for 48 h, the AgNPs were collected and the negative surface charge and size distribution of the synthesized AgNPs were measured using dynamic light scattering (DLS). The AgNP structure was determined by high-resolution powder X-ray diffraction (XRD) and the AgNP morphology was investigated using transmission electron microscopy (TEM). AgNP antibacterial activities were evaluated against Bacillus cereus, Staphylococcus aureus, Escherichia coli, Salmonella enteritidis, and Pseudomonas aeruginosa using the disc diffusion method. Additionally, minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values were also determined. Biosynthesized AgNPs showed enhanced antibacterial activity against B. cereus, S. aureus, E. coli, S. enteritidis, and P. aeruginosa compared with that of pristine solvent extract. These results suggest that AgNPs synthesized from extracts of DI are promising antibacterial agents against pathogenic bacteria and can be further applied in the food industry.

Network Pharmacology Combined with Experimental Validation Reveals the Anti-tumor Effect of Duchesnea indica against Hepatocellular Carcinoma.

Context: Duchesnea indica is effective against hepatocellular carcinoma (HCC); however, its underlying mechanism of action remains unclear. Objective: The present study aimed to investigate the potential mechanism of action and effects of D. indica components against HCC. Materials and methods: First, the effects of D. indica against HCC were investigated in vitro and in vivo. For in vitro experiments, HCC cell lines were treated with D. indica solutions at different concentrations (0, 1, 2 mg/mL) and then assessed for cell apoptosis, proliferation, migration, invasion, and angiogenic ability. For in vivo experiments, 24 mice were randomly divided into the following four groups: model group and D. indica low-, medium-, and high-dose groups. Tumor growth and CD34 and Ki67 expression levels were assessed to determine the effects of D. indica on cell proliferation and angiogenic ability. Furthermore, transcriptome sequencing and differential expression analyses were used to identify D. indica-induced differentially expressed genes (DEGs) in HCC cells. Additionally, mass spectrometry was conducted to identify the chemical components of D. indica. Four databases were used to predict the target proteins of these chemical components in HCC. HCC-associated genes were identified from two databases. By intersecting the identified DEGs; target proteins; and HCC-associated genes, key D. indica-regulated HCC-related genes were identified. Subsequently, protein-protein interaction network, network pharmacology, and molecular docking were used to identify the active compounds in D. indica and their likely gene targets. Results: In vitro experiments demonstrated that D. indica induced tumor cell apoptosis and inhibited cell proliferation, migration, invasion, and angiogenic potential. In vivo experiments demonstrated that D. indica inhibited tumor growth in a dose-dependent manner. Bioinformatic analyses identified 49 key D. indica-regulated HCC-related genes, of which FOS, SERPINE1, AKR1C3, and FGF2 were the most significant. Mass spectrometry identified the following five molecules in D. indica with potential anti-HCC activity: 4', 5, 7-trihydroxyflavone; ethyl protocatechuate; 3, 5-dihydroxy-benzoic acid; curculigosaponin A; and curculigine G. Molecular docking validated the interaction between D. indica active compounds and their target proteins in HCC. Conclusions: The present study confirmed the therapeutic effects of D. indica against HCC and identified the key genes and active components that may contribute to its mechanism of action, thereby providing a basis for further research on targeted therapeutics for HCC.

Antimicrobial Activity of Chitosan/Gelatin/Poly(vinyl alcohol) Ternary Blend Film Incorporated with Duchesnea indica Extract in Strawberry Applications.

Chitosan (CTS)/gelatin (GEL)/poly(vinyl alcohol) (PVA)-based composite films with different concentrations of Duchesnea indica extract (DIE) (6.25 and 25 mg/mL), an antimicrobial agent, were manufactured using a casting technique. Results indicated that elongation at break decreased as DIE was added at higher concentrations. Composite films showed no significant differences in thickness, tensile strength, and water vapor permeability. Scanning electron microscopy images revealed that DIE was successfully incorporated into film matrices to interact with polymers. The addition of DIE to the film inhibited the growth of S. aureus by up to 4.9 log CFU/mL. The inhibitory effect on S. aureus using DIE-incorporated coating applied to strawberries was greatest at room temperature storage for 24 h only when it was coated twice or more. The maximum inhibition in strawberries was 2.5 log CFU/g when they were coated twice and 3.2 log CFU/g when they were coated three times. The results of this study suggest that DIE could be used as a natural antimicrobial agent, and DIE-integrated CTS/GEL/PVA films or coatings have potential as a food packaging alternative for preventing foodborne pathogen contamination.

Anxiolytic and antidepressant potential of extracts of Duchesnea Indica in animal models.

THIS study was designed to investigate the anxiolytic and antidepressant activity of Duchesnea Indica. The methanolic extract and n-hexane fraction of plant were tested in albino mice (20-30 g of 6-8 week). Safety profile of each extract was determined at different doses. Anxiolytic effect of plant was determined by Elevated Plus Maze and Light and Dark Model at different doses of methanolic and n-hexane extract. Antidepressant activities were assisted by Tail Suspension and Forced Swim Model at different doses. Result illustrated that D. Indica had a significant (P < 0.05) potential of reducing anxiety and depression. The methanolic extract at 100 mg and 200 mg significantly increased the time spend in light region of light and dark model and more time spent in open arm of Elevated Plus Maze model. n-hexane extract at dose of 5 mg and 10 mg/kg significantly increases the time spend in light region of Light and dark model and more time spend in open arm of Elevated Plus Maze model as compare to Control group. Methanolic extract of D. Indica significantly reduced the time of immobility in Tail Suspension and Force Swim Model at Dose of 100 mg and 200mg/kg. n-hexane extract also exhibit anti-depressant effect by reducing the time of immobility in Force swim and tail suspension Model at 100 mg and 200mg/kg dose as compare to control group. From the above observations, it could be assumed that the plant has marked anxiolytic and antidepressant potential. However further additional studies will be necessary to determine the underlying exact mechanism and clinical uses.

Rapid identification and isolation of neuraminidase inhibitors from mockstrawberry (Duchesnea indica Andr.) based on ligand fishing combined with HR-ESI-Q-TOF-MS.

Neuraminidase inhibitors (NAIs) are the mainstay antiviral drugs against influenza infection. In this study, a ligand fishing protocol was developed to screen NAIs using neuraminidase immobilized magnetic beads (NA-MB). After verifying the feasibility of NA-MB with an artificial mixture including NA inhibitors and non-inhibitors, the developed ligand fishing protocol was applied to screen NAIs from the crude extracts of Duchesnea indica Andr. Twenty-four NA binding compounds were identified from the normal butanol (n-BuOH) extract of D. indica as potential NAIs by high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (HPLC-Q-TOF-MS) assisted with Compound Structure Identification (CSI):FingerID, including 12 ellagitannins, 4 brevifolin derivatives, 3 ellagic acid derivatives, and 4 flavonoids. Among them, 9 compounds were isolated and tested for in vitro NA inhibitory activities against NA from Clostridium perfringens, and from oseltamivir sensitive and resistant influenza A virus strains. The results indicate that compound B23 has the NA inhibitory activities in both the oseltamivir sensitive and resistant viral NA, with half maximal inhibitory concentration (IC50) values of 197.9 and 125.4 µmol/L, respectively. Moreover, B23 can obviously reduce the replication of oseltamivir sensitive and resistant viruses in Madin-Darby canine kidney (MDCK) cells at the concentrations of 40 and 200 µmol/L.

Rhizospheric soil fungal community patterns of Duchesnea indica in response to altitude gradient in Yunnan, southwest China.

The magnitude of the impact of altitude gradient on microbial community and diversity has been studied in recent decades. Whereas bacteria have been the focus of most studies, fungi have been given relatively less attention. As a vital part of the macro- and microscopic ecosystem, rhizosphere fungi play a key role in organic matter decomposition and relative abundance of plant species and have an impact on plant growth and development. Using Duchesnea indica as the host plant, we examined the rhizosphere soil fungal community patterns across the altitude gradient in 15 sites of Yunnan province by sequencing the fungal ITS2 region with the Illumina MiSeq platform. We determined the fungal community composition and structure. We found that, surprisingly, rhizosphere soil fungal diversity of D. indica increased with altitudinal gradient. There was a slight difference in diversity between samples from high- and medium-altitude sites, with medium-altitude sites having the greater diversity. Furthermore, the rhizosphere soil fungal community composition and structure kept changing along the altitudinal gradient. Taxonomic results showed that the extent of phylum diversity was greatest at high-altitude sites, with Ascomycota, Basidiomycota, Zygomycota, and Glomeromycota as the most dominant fungal phyla.

Characterization, antioxidant, and anticancer activities of a neutral polysaccharide from Duchesnea indica (Andr.) Focke.

A neutral polysaccharide (DIP-1) from Duchesnea indica (Andr.) Focke was obtained by hot water extraction, ethanol precipitation and chromatographic separation (DEAE-52 cellulose anion-exchange column and Sephadex G-100 gel column). The physicochemical properties of DIP-1 were elucidated by gel permeation chromatography, monosaccharide composition, Fourier transform infrared spectrometry, UV-visible spectrophotometry, scanning electron microscope and Congo red test. The results indicated that DIP-1 was consisted of mannose, glucosamine, glucose, galactose and arabinose in a ratio of 1.00:0.42:18.36:14.17:0.81, and its molecular weight was 218.3 kDa. Meanwhile, DIP-1 presented a straight hexahedron structure, but no triple-helical conformation. In antioxidant activity tests, DIP-1 exhibited powerful scavenging activities on hydroxyl, DPPH, ABTS radicals and reducing power in a dose-dependent manner. Especially, DIP-1 demonstrated high inhibitory activities against SKOV-3 and Hep-G2 cells in vitro, with IC50 values of 1.42 and 1.23 mg/ml, respectively. PRACTICAL APPLICATIONS: D. indica has been used for a long time as a Chinese medicine for therapy of many diseases, including cancer, inflammation, leprosy, fever, bleeding and so on. At present, polysaccharides have attracted comprehensive attention because of a large range of pharmacological and biological properties, including antitumor, antidiabetic, antioxidant and immunomodulatory activity. In the present study, we purified and characterized a neutral polysaccharide from D. indica for the first time. Moreover, the neutral polysaccharide exhibits significant antioxidant and antitumor activities. Therefore, the present study laid a foundation for the high-value application of D. indica polysaccharides in functional food and pharmaceutical industries.

Duchesnea indica extract attenuates oral cancer cells metastatic potential through the inhibition of the matrix metalloproteinase-2 activity by down-regulating the MEK/ERK pathway.

BACKGROUND: Duchesnea indica (Andr.) Focke, an herb in folk medicine used extensively in traditional Chinese medicine, has cytostatic properties as well as antioxidant and antimetastasis activities in various cancer cells. However, the effects and underlying mechanisms of Duchesnea indica extracts (DIEs) on human oral squamous cell carcinoma (OSCC) metastases remain unclear. PURPOSE: In this study, we posit the hypothesis that DIE possesses antimetastatic effects on human OSCC cells. METHODS: The effects of DIE on cell viability, motility, migration, and invasion were investigated. Gelatin zymography, Western blotting, migration and invasion assays were used to further study the underlying mechanisms involved in the antimetastatic effects of DIE in OSCC cells. RESULTS: The results from MTT assay revealed that DIE did not affect the cell viability of OSCC cells. Moreover, DIE significantly attenuated OSCC cells' motility, migration, and invasion by reducing the MMP-2 protein expression and MMP-2 activity in a dose-dependent manner. In addition, DIE reduced the phosphorylation of both ERK1/2 and its upstream kinase but had no effect on the phosphorylation of p38 and JNK. CONCLUSION: DIE triggers the antimetastatic activity in OSCC cells by suppressing the MMP-2 activity via the MEK/ERK signaling pathways. Therefore, these findings are promising for the use of DIE antimetastatic activity in oral cancer metastasis treatment.

Optimization of extraction technology and stability of anthocyanins from Duchesnea indica by response surface methodology / 中草药

Objective To optimize extraction technology and evaluate the stability of anthocyanins from Duchesnea indica by response surface methodology. Methods Based on single factor experiment, the ethanol concentration, liquid-material ratio, extraction time, and extraction temperature were selected as independent variables, and the anthocyanin extraction ratio was selected as response value. The Box-Behnken design method and response surface methodology were used to optimize the extraction technology. Additionally, the effects of pH, temperature, organic acid, and oxidizer on the stability of anthocyanins were investigated. Results The optimized extraction technology: ethanol concentration was 80%, liquid-material ratio was 10:1, extraction time was 93 min, and extraction temperature was 36 ℃. The ratio of the extraction of anthocyanins with the optimal technology was 1.126%. The stability experiments results showed that the anthocyanins should keep in acid and low temperature conditions. Adding citric acid and gallic acid enhanced its stability, but glycine and H2O2 decreased stability. Conclusion The extraction technology optimized by response surface methodology was scientific, reasonable, and feasible, and it can be applicable for the extraction of anthocyanins from D. indica. Moreover, the condition should be paid attention to in order to maintain the stability of anthocyanins from D. indica.

Duchesnea indica extract suppresses the migration of human lung adenocarcinoma cells by inhibiting epithelial-mesenchymal transition.

Epithelial-mesenchymal transition (EMT) is a process through which epithelial cells are transformed into mesenchymal cells; EMT diminishes cell polarity and cell-cell adhesion in cancer cells, leading to enhanced migratory and invasive properties. In this experiment, zymography, cell invasion, and migration assays were performed. Results indicated that Duchesnea indica extracts (DIE) inhibited highly metastatic A549 and H1299 cells by reducing the secretions of matrix metalloproteinase-2 and urokinase-type plasminogen activator. Cell adhesion assay also demonstrated that DIE reduced the cell adhesion properties. Western blot analysis showed that DIE down-regulated the expression of N-cadherin, fibronectin, and vimentin, which are mesenchymal markers, and enhanced that of E-cadherin, which is an epithelial marker. In vivo study showed that tumor growth was significantly reduced in BALB/c nude mouse xenograft model administered with oral gavage of DIE. Therefore, DIE could be exhibits potential as a phytochemical-based platform for prevention and treatment of lung cancer. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 2053-2063, 2017.

Simultaneous determination of five flavonoids in Duchesnea indica at five picking time by HPLC / 中成药

AIM To establish an HPLC method for the simuhaneous content determination of five flavonoids in Duchesnea indica (Andr.) Focke at five picking time (April,May,June,July and August).METHODS The analysis of D.indica methanol extract was performed on a 25 ℃ thermostatic Agilent ZORBAX SB-C18 column (250 mm×4.6 mm,5 μm),the mobile phase comprising of acetointrile-0.1％ methanoic acid flowing at 1.0 mL/min in a gradient elution manner,and the detection wavelength was set at 350 nm.RESULTS Rutin,hyperoside,isoquercitrin,celereoin and kaempferol showed good linear relationships within their own ranges (R2 ≥0.998 6),whose average recoveries were 97.1％-101.5％ with the RSDs of 1.37％-2.37％.The contents of five constituents in samples at different picking time exhibited obvious differences,among which lutin and hyperoside contents were the highest in June,isoquercitrin content was the highest in July,and celereoin and kaempferol contents were the highest in August.CONCLUSION The suitable picking time of D.indica is June and July.

Phenolic Profiling of Duchesnea indica Combining Macroporous Resin Chromatography (MRC) with HPLC-ESI-MS/MS and ESI-IT-MS.

Duchesnea indica (D. indica) is an important traditional Chinese medicine, and has long been clinically used to treat cancer in Asian countries. It has been described previously as a rich source of phenolic compounds with a broad array of diversified structures, which are the major active ingredients. However, an accurate and complete phenolic profiling has not been determined yet. In the present work, the total phenolic compounds in crude extracts from D. indica were enriched and fractionated over a macroporous resin column, then identified by HPLC-ESI-MS/MS and ESI-IT-MS (ion trap MS). A total of 27 phenolic compounds were identified in D. indica, of which 21 compounds were identified for the first time. These 27 phenolic compounds encompassing four phenolic groups, including ellagitannins, ellagic acid and ellagic acid glycosides, hydroxybenzoic acid and hydroxycinnamic acid derivatives, and flavonols, were then successfully quantified using peak areas against those of the corresponding standards with good linearity (R² > 0.998) in the range of the tested concentrations. As a result, the contents of individual phenolic compounds varied from 6.69 mg per 100 g dry weight (DW) for ellagic acid to 71.36 mg per 100 g DW for brevifolin carboxylate. Not only did this study provide the first phenolic profiling of D. indica, but both the qualitative identification and the subsequent quantitative analysis of 27 phenolic compounds from D. indica should provide a good basis for future exploration of this valuable medicinal plant.

Extraction of crude polysaccharides from Duchesnea indica (Andrews) Focke: optimization by response surface methodology.

A full set of optimization procedure was applied to the extraction of anti-viral polysaccharides from Duchesnea indica (Andrews) Focke. By Plackett-Burman factorial design, three parameters (extraction time, extraction temperature, and ratio of water to raw material) were identified as significant to the extraction yield. However, no significant parameters had been identified for antiviral activity. A three-level-three-factor Box-Behnken factorial design was then employed to further optimize the extraction condition. The experimental data were fitted to a second-order polynomial equation using multiple regression analysis and also examined using appropriate statistical methods. This led to the construction of a response surface indicating the optimal values for each parameter and response studied. Concerning the extraction yield, an extraction at 98.51 ºC for 6.16 h with a ratio of water to raw material of 30.94 mL/g was found to be optimal. Under the optimized conditions, the experimental yield was 6.430 ± 0.078%, which was well matched with the predicted yield of 6.509%.

Analysis of the Genetic Diversity of Germplasm Resources of Duchesnea indica from Hunan by Random Amplified Polymorphic DNA / 中国药房

OBJECTIVE:To study the genetic diversity of germplasm resources of Duchesnea indica from Hunan. METHODS:The random amplified polymorphic DNA(RAPD)reaction system of germplasm resources was established and RAPD-PCR was ad-opted to detect the expressions of 24 groups of D. indica from Hunan(including 21 wild and 3 cultivated varietos in different geo-graphical distribution). Agarose gel electrophoresis was used to analyze the results and calculate allele number (Na),effective al-lele number (Ne) the polymorphism points percentage (PPB),Nei’s gene diversity index (H) and Shannon’s information index (I). RESULTS:Totally 14 polymorphic primers were screened and 90 electrophoresis bands were amplified,including 81 polymor-phic bands with Na of 1.900 0,Ne of 1.540 1,PPB of 90.0%,H of 0.312 8 and I of 0.467 5. CONCLUSIONS:The genetic diver-sity of D. indica from Hunan is rich,and there were differences in genetic background between the cultivated species and wild spe-cies;clustering results show significant correlation with geographic distribution.

Analysis of Volatile Components in Duchesnea Indica by Different Extraction Methods / 分析化学

ThevolatilecompositionsofDuchesneaindicawerestudiedbyheadspacesolid-phase microextraction ( HS-SPME ) , soxhlet extraction ( SE ) , ultrasonic assistant extraction ( UAE ) and steam distillation ( SD) coupled with gas chromatography-mass spectrometry ( GC-MS) . The experimental parameters of HS-SPME, including fiber type, extraction temperature, extraction time and desorption time were investigated. 47, 32, 16 and 16 compounds were identified by HS-SPME, SD, SE and UAE extracting methods, respectively. 66 compounds were obtained in total, among which 47 compounds were first reported in Duchesnea indica. The experimental results showed that terpenoids were the most abundant compositions in HS-SPME and SD, but acids accounted for 61. 44% and 69. 54% of the total content obtained by SE and UAE.

Study on chemical constituents of Duchesnea indica Andr. Focke / 第二军医大学学报

Objective: To investigate the chemical constituents in the EtOAc fraction of the EtOH extract of Duchesnea indica Andr. Focke. Methods: Extraction was done with 95% EtOH. EtOAc fraction of the EtOH extract was isolated and purified by column chromatography using silica gel,RP-C18,Sephadex LH-20 and HPLC. The structures of the compounds were identified by their physico-chemical properties and spectral analysis. Results: Thirteen compounds were obtained from the EtOAc fraction of Duchesnea indica Andr. Focke and they were identified as euscaphic acid(1),arjunic acid(2),p-hydroxy cinnamic acid (3),apigenin(4),kaempferol(5),2α-hydroxy ursolic acid(6),2α-hydroxy oleanolic acid(7),tomentic acid(8),kaii-ichigeside F1 (9), potengriffioside A (10), rosamultin (11), kaempferol-3-O-β-D-glucoside (12), and isoquercitrin (13). Conclusion: The compounds 2-5,10, and 12 have been isolated from the genus of Duchesnea for the first time.