Comparative proteomic analysis of edible bird's nest from different origins.

Edible bird's nest (EBN) mainly made of saliva that secreted by a variety of swiftlets is a kind of precious traditional Chinese medicine. EBNs from different biological and geographical origins exhibit varieties in morphology, material composition, nutritive value and commercial value. Here, we collected four different EBN samples from Huaiji, China (Grass EBN), Nha Trang, Vietnam (Imperial EBN) and East Kalimantan, Indonesia (White EBN and Feather EBN) respectively, and applied label-free quantitative MS-based proteomics technique to identify its protein composition. First, phylogenetic analysis was performed based on cytb gene to identify its biological origin. Second, a total of 37 proteins of EBNs were identified, among which there were six common proteins that detected in all samples and exhibited relatively higher content. Gene ontology analysis revealed the possible function of EBN proteins, and principal component analysis and hierarchical clustering analysis based on 37 proteins were performed to compare the difference of various EBNs. In summary, our study deciphered the common and characteristic protein components of EBNs of different origins and described their possible functions by GO enrichment analysis, which helps to establish an objective and reliable quality evaluation system.

Characteristic quality analysis for biologically induced agarwood columns in Aquilaria sinensis.

Current artificial agarwood-inducing techniques yield low quality and quantities of agarwood. On account of unclear agarwood formation mechanism there's still no high-efficiency agarwood inducing method globally spread. In this study, a complete agarwood column was taken out of the live tree trunk at 6 months post-treatment by a novel agarwood-inducing method (Agar-Bit) in cultivated Aquilaria sinensis trees, and was first divided into 8 parts (A1-4, B1-4) involving agarwood layer (A part) and brown inner layer (B part) according to its color and length for analysis. These eight parts were analyzed microscope observation, 6 chromones' contents and characteristic chromatograms by HPLC (high performance liquid chromatography), GC-MS (gas chromatography-mass spectrometer) with to determine chemical changes. Other quality characteristics, TLC (thin-layer chromatography) and alcohol soluble extraction content, were also determined. Our results showed that resin changed with A to B part and microstructure changed with length. Six chromones in the eight parts varied with layers. Result of characteristic chromatograms showed that both A and B parts contained six characteristic peaks. Volatile component distributed mainly in A part, but important chromones were also detected in B parts. Results from TLC and alcohol soluble extraction content also showed that B part contained characteristic compounds of agarwood. In addition, some compounds in the essential oil detected by GC-MS in A part produced by Agar-Bit were similar to that found in natural agarwood, compounds in B parts were similar to BC agarwood, as were the results for the TLC and alcohol soluble extraction content. In conclusion, the chemical distribution obtained here from Agar-Bit could provide some clues to optimize high production and high efficiency stimulating method for whole tree full of resin in Aquilaria sinensis and to reveal the subtle agarwood formation mechanism throughout a whole trunk.

Covalent Modification of Proteins by Osthole Reactive Metabolites using Proteomic Approaches.

BACKGROUND: Osthole (OST) is a bioactive natural coumarin derived from the plant Cnidium monnieri (L.) Cusson fruit (She Chuang Zi), which has various pharmacological and biological activities. OST contains an α,ß- unsaturated lactone, which is an electrophilic group that tends to be metabolized into reactive metabolites (RMs). Then, RMs are able to covalently modify nucleophilic amino acid (AA) residues of target proteins. However, few researchers considered the contribution of the covalent modification induced by OST or its metabolites. OBJECTIVE: This study aims to investigate the metabolic profile and the metabolites-protein modification of OST. METHODS: The metabolites of OST were qualitatively identified using UHPLC-Q-TOF-MS. The RMs modification patterns and potentially modified AA residues were confirmed by UHPLC-Q-TOF-MS using rat liver microsomes (RLMs) and model AAs. Finally, the modified peptides derived from high-abundance microsomal peptides were separated via nano-LC-Orbitrap-MS, and then RM-modified proteins were identified using a proteome discoverer. RESULTS: In the presence of RLMs, OST could rapidly be metabolized within 1 h and hardly identified at 4 h. We detected 10 OST metabolites, 13 OST metabolites-NAC (N-acetyl cysteine) adducts, 3 NAL (N-acetyl lysine) adducts, and 11 GSH (glutathione) adducts. Furthermore, 16 RM-modified protein targets were identified, many of which are included in the essential biological processes of OST's anti-Alzheimer's disease (AD) and anti-tumor. CONCLUSION: This study provides a novel perspective on the molecular mechanism of OST's pharmacological activities, as well as identifies potential targets for further development and application of OST and other Natural products (NPs).

Combination of chemical profiling and network pharmacology analysis to investigate the potential mechanism of Li-Zhong-Xiao-Pi granules in the treatment of gastric precancerous lesions.

Li-Zhong-Xiao-Pi granules (LZXP) are effective for treating gastric precancerous lesions (GPL) in traditional Chinese medicine. However, the active compounds of LZXP and their potential therapeutic mechanism in GPL remained unclarified. The purpose of this study is to investigate the chemical composition and potential targets of LZXP. Based on the accurate masses, ion fragments, and literature data, a total of 128 compounds were identified in the LZXP sample using ultra-performance liquid chromatography and quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) in both positive and negative ion modes, and 28 of these compounds were exactly determined by comparison with authentic reference standards. Meanwhile, 11 typical components were quantified via UPLC during a 24 min period. The linearity, accuracy, stability and recovery of the method were all proven. Through the network pharmacological analysis, six chemicals (quercetin, 4'-hydroxywogonin, sinensetin, 5, 7, 8, 3', 4'-pentamethoxyflavanone, 8-gingerdione and quercetin) were identified as the active ingredients, and five LZXP targets (AKT1, CYP1B1, PTGS2, MMP9 and EGFR) were found to be the crucial molecules in the treatment of GPL. This study provides a systematic and applicable method for the rapid screening and identification of the chemical constituents from LZXP, and an effective understanding the mechanism of LZXP in the treatment of GPL.

Physcion, a novel inhibitor of 5α-reductase that promotes hair growth in vitro and in vivo.

Androgenic alopecia (AGA) has a high incidence. Excess dihydrotestosterone in blood capillaries, which is converted from testosterone by 5α-reductase, is an AGA causative factor. We identified the inhibitory activity of four Polygonum multiflorum compounds against 5α-reductase via high-performance liquid chromatography, and the results showed that Physcion was a potent 5α-reductase inhibitor. Additionally, we found that through inhibiting 5α-reductase expression, Physcion could shorten the time of dorsal skin darkening and hair growth, improve hair follicle morphology, and significantly increase hair follicle count. Eventually, through molecular docking study, we found the binding energy and molecular interactions between Physcion and 5α-reductase type II. These results suggested that Physcion is a potent 5α-reductase inhibitor, as well as a new natural medicine for treating AGA.

Genetic Identification of Edible Bird's Nest in Thailand Based on ARMS-PCR.

Edible bird's nest (EBN) is a popular delicacy in the Asian Pacific region originating from Indonesia, Malaysia, Thailand and Vietnam, which consist of various potential medicine value in Traditional Chinese Medicine (TCM). Thailand is one of the main exporters of EBN. However, the genetic information of EBN, a key part of molecular biology, has yet to be reported in Thailand. It is necessary to explore the genetic information of EBN in Thailand based on a quick and simple method to help protect the rights and interests of consumers. This research aimed to systematically evaluate different methods of extracting EBN DNA to improve the efficiency of the analysis of cytochrome b (Cytb) and NADH dehydrogenase subunit 2 (ND2) gene sequences, the establishment of phylogenetic trees, and the genetic information of EBN in Thailand. Additionally, we aimed to develop a quick and simple method for identifying EBN from different species based on the genetic information and amplification-refractory mutation system PCR (ARMS-PCR). By comparing the four methods [cetyltrimethylammonium bromide (CTAB), sodium dodecyl sulfate (SDS), kit and guanidinium isothiocyanate methods] for EBN extraction, we found that the guanidinium isothiocyanate method was the optimal extraction method. Phylogenetic trees generated on the basis of Cytb and ND2 gene analyses showed that 26 samples of house EBN and 4 samples of cave EBN came from Aerodramus fuciphagus and Aerodramus maximus, respectively. In addition, to distinguish different samples from different species of Apodiformes, we designed 4 polymerase chain reaction (PCR) amplification primers based on the ND2 gene sequences of A. fuciphagus and A. maximus. The ARMS-PCR results showed band lengths for A. fuciphagus EBN of 533, 402, and 201 bp, while those for A. maximus EBN were 463, 317, and 201 bp. Collectively, the results showed that ARMS-PCR is a fast and simple method for the genetic identification of EBN based on designing specific original identification primers.

Diagnostic Performance of Diffusion MRI for differentiating Benign and Malignant Nonfatty Musculoskeletal Soft Tissue Tumors: A Systematic Review and Meta-analysis.

Objective: To evaluate the diagnostic performance of standard diffusion-weighted imaging (DWI), intravoxel incoherent motion (IVIM), and diffusion kurtosis imaging (DKI), for differentiating benign and malignant soft tissue tumors (STTs). Materials and methods: A thorough search was carried out to identify suitable studies published up to September 2020. The quality of the studies involved was evaluated using Quality Assessment of Diagnostic Accuracy Studies-2 (QUADAS-2). The pooled sensitivity (SEN), specificity (SPE), and summary receiver operating characteristic (SROC) curve were calculated using bivariate mixed effects models. A subgroup analysis was also performed to explore the heterogeneity. Results: Eighteen studies investigating 1319 patients with musculoskeletal STTs (malignant, n=623; benign, n=696) were enrolled. Thirteen standard DWI studies using the apparent diffusion coefficient (ADC) showed that the pooled SEN and SPE of ADC were 0.80 (95% CI: 0.77-0.82) and 0.63 (95% CI: 0.60-0.67), respectively. The area under the curve (AUC) calculated from the SROC curve was 0.806. The subgroup analysis indicated that the percentage of myxoid malignant tumors, magnet strength, study design, and ROI placement were significant factors affecting heterogeneity. Four IVIM studies showed that the AUCs calculated from the SROC curves of the parameters ADC and D were 0.859 and 0.874, respectively. The AUCs for the IVIM parameters pseudo diffusion coefficient (D*) and perfusion fraction (f) calculated from the SROC curve were 0.736 and 0.573, respectively. Two DKI studies showed that the AUCs of the DKI parameter mean kurtosis (MK) were 0.97 and 0.89, respectively. Conclusion: The DWI-derived ADC value and the IVIM DWI-derived D value might be accurate tools for discriminating musculoskeletal STTs, especially for non-myxoid SSTs, using more than two b values, with maximal b value ranging from 600 to 800 s/mm2, additionally, a high-field strength (3.0 T) optimizes the diagnostic performance.

Molecular analysis of edible bird's nest and rapid authentication of Aerodramus fuciphagus from its subspecies by PCR-RFLP based on the cytb gene.

Edible bird's nest (EBN), for its great nutritional value, is widely used around the world, especially in China and Singapore. EBNs of different origins and types may vary in price and quality. Nowadays, birds' nests are difficult to identify morphologically, except for some whole bird's nests of which origins can be roughly identified. In this study, forty-two samples were collected from different regions for sequencing analysis and phylogenetic classification to initially determine their origins. Two stable enzyme digestion sites were found in the analysis of restriction maps of the species. Then, a quick and specific PCR-RFLP method was established to identify the EBN samples' origins. The genetic identification results indicated that the forty-two samples were from five origins. With the Af/g-486bp-F/R primer and restriction enzyme Taq I, Aerodramus fuciphagus (A. fuciphagus) was efficiently differentiated from the other species. Furthermore, the cytb-592bp-F/R primer and the BamH I enzyme were found to be useful in distinguishing Aerodramus fuciphagus (A. fuciphagus) from its subspecies (Aerodramus germani, A. germani). The PCR-RFLP method provides a potential tool for the rapid discrimination of A. fuciphagus at the species and even the subspecies levels to ensure the quality of the EBN products.

Antiviral activity of lycorine against Zika virus in vivo and in vitro.

The emergence and re-emergence of Zika virus (ZIKV), is a cause for international concern. These highly pathogenic arboviruses represent a serious health burden in tropical and subtropical areas worldwide. Despite these burdens, antiviral therapies do not exist, and inhibitors of ZIKV are therefore urgently needed. To elucidate the anti-ZIKV effect of lycorine, we used reverse transcription-quantitative real-time PCR (qRT-PCR), immunofluorescence, Westernwestern blot, and plaque forming assay to analyse viral RNA (vRNA), viral protein, progeny virus counts, and validated inhibitors in vitro using a variety of cell lines. Additionally, we found that lycorine acts post-infection according to time-of-addition assay, and inhibits RdRp activity. Lycorine protected AG6 mice against ZIKV-induced lethality by decreasing the viral load in the blood. Due to its potency and ability to target ZIKV infection in vivo and in vitro, lycorine might offer promising therapeutic possibilities for combatting ZIKV infections in the future.

[Clinical trials of treatment of post-stroke foot drop by fire-needling plus stuck-needle-stretching].

OBJECTIVE: To compare the therapeutic effect of fire-needling plus stuck-needle-stretching and conventional acupuncture for post-stroke foot drop, so as to provide a reference foundation for optimizing clinical treatment regimen. METHODS: A total of 60 patients with post-stroke foot drop were equally randomized into a treatment group and a control group. In the treatment group, fire-needling plus stuck-needle-stretching was applied to acupoint pairs Jiexi (ST41)-Taichong (LR3), Xiajuxu (ST39)-ST41, Fenglong (ST40)-ST39, shangjuxu (ST37)-ST40, Zusanli (ST36)-ST37, Dubi (ST35)-ST36, Xuanzhong (GB39)-Qiuxu (GB40) on the affected side, and Chengshan (BL57) and Ashi points were swiftly pricked with red-hot filiform needle without needle retaining. In the conventional acupuncture group, the same acupoints were needled with filiform needles, and the treatment for both groups was given once a day, 6 times a week, for consecutive 4 weeks. The spasm severity of posterior triceps of the lower leg was evaluated using modified Ashworth scale, the tibial anterior muscle strength was measured using Lovett' and Martin's methods, the activities of daily living were assessed using modified Barthel's index, the walking ability was evaluated using Holden walking rating scale and the lower limb motor function assessed using Fugl-Meyer assessment scale, and the severity of foot drop was assessed in reference to Garceau criteria. RESULTS: After the treatment, the score of the modified Ashworth scale was significantly reduced (P<0.01), and those of Lovett muscle strength scale, Barthel index, Holden walking rating scale and Fugl-Meyer lower limb motor function scale were considerably increased in both groups versus their own pre-treatment (P<0.01). The therapeutic effect of the treatment group was significantly superior to that of the control group in reducing Ashworth scale score and in increasing the scores of Lovett muscle strength test, Barthel index, Holden walking function scale and Fugl-Meyer lower limb motor function scale (P<0.01). Of the two 30 cases in the control and treatment groups, 10(33.33%)and 14(46.67%) experienced a remarkable improvement, 10(33.33%)and 12(40.00%)were improved, 7(23.33%)and 3(10.00%)had a mild progress, 3(10.00%) and 1(3.33%) failed, with the excellent plus good effective rates being 66.67% and 86.67%, respectively, but without significant difference between the two groups (P>0.05). CONCLUSION: Both fire-needling plus stuck-needle-stretching and conventional acupuncture are effective in the treatment of post-stroke foot drop, but the former is relatively better.

Bioactive glycosides from the roots of Ilex asprella.

Context The roots of Ilex asprella (Hook. et Arn.) Champ. ex Benth. (Aquifoliaceae) are widely used in Chinese medicine to treat influenza, amygdalitis, pertussis, etc. Their mechanism of action is still unknown, which raises the need to identify new bioactive compounds in this plant. Objective In this study, we isolated a novel saponin containing sulphonic groups, namely, asprellcoside A (1) and a known phenolic glycoside compound (2) from the roots of Ilex asprella and evaluated their bioactivities. Materials and methods Molecular structures were elucidated by analysing their spectral and chemical properties. The viability of A549 cells was tested using a MTT assay. Ability of the compounds to inhibit viruses was determined using the neuraminidase activity assay. Their anti-inflammatory effects were tested using the IP-10 activity assay using various concentrations (compound 1: 0.6, 0.2, 0.6, 1.70, 5.00 and 15.00 µM; compound 2: 0.4, 1.2, 3.6, 11.0, 33.0 and 100 µM). Their inhibitory effect on platelet aggregation induced by adenosine diphosphate (ADP) in rabbit plasma was determined at 60 and 80 µM. Results Both compounds inhibit influenza virus strain A/PuertoRico/8/1934 (H1N1) strongly with EC50 values of 4.1 and 1.7 µM, respectively. Both compounds inhibit the secretion of IP-10 with EC50 values of 6.6 and 2.5 µM, respectively. Compound 1 alone inhibited platelet aggregation significantly, with the rate of suppression being 47 ± 8 and 38 ± 3%, at 60 and 80 µM, respectively. Conclusions The results suggest that both compounds may be valid therapeutics against influenza virus infection and that compound 1 may be a novel agent for treating thrombosis.

Inhibitory activities of some traditional Chinese herbs against testosterone 5α-reductase and effects of Cacumen platycladi on hair re-growth in testosterone-treated mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Many traditional Chinese medicines (TCM) have been used for hundreds of years for hair blackening and hair nourishing, and now many of them are commonly used in Chinese herbal shampoo to nourish the hair and promote hair growth. AIMS OF THE STUDY: The present study was performed to screen 5α-reductase (5αR) inhibitors from traditional Chinese medicines, evaluate its hair growth promoting activity in vivo, and further investigate its effects on androgen metabolism and the expression of 5αR II in hair follicles. MATERIALS AND METHODS: Nine TCM which were dried, ground and extracted by maceration with 75% ethanol or distilled water were used for screening 5αR inhibitors, and enzymes were extracted from the rat epididymis. The leaves of Platycladus orientalis (L.) Franco was used to evaluate the in vivo anti-androgenic activity. Skin color was observed daily and the hair re-growth was assessed by assigning the hair growth score. The longitudinal sections of hair follicles were used for observing follicle morphology, classifying of distinct stages of hair follicle morphogenesis and calculate the average score. The transverse sections were used for determination of hair follicle counts. Testosterone (T), Dihydrotestosterone (DHT) and Estradiol (E2) levels in serum and skin tissue were detected by ELISA kits. The immunofluorescence assay was used to detect the influence of CP-ext on 5αR expression in dorsal skin. RESULTS: We found the extract of Ganoderma lucidum (GL-ext), Polygonum multiflori (PM-ext), Cacumen platycladi (CP-ext) and Cynomorium songaricum (CS-ext) showed stronger 5αR inhibitory activity. CP-ext (5mg and 2mg/mouse/day) could significantly shorten the time of the dorsal skin darkening and got longhaired (P<0.01), and showed high hair re-growth promoting activity. Furthermore the histological data of hair follicles in each group showed that CP-ext could promote the growth of hair follicle and slowed down hair follicles enter the telogen. What's more CP-ext significantly reduced DHT levels and down-regulated the expression of 5αRⅡin skin (P<0.01). CONCLUSIONS: GL-ext, PM-ext, CP-ext and CS-ext showed strong 5αR inhibitory activity. CP-ext possesses high hair growth promoting activity in the in vivo androgen-sensitive mouse model via inhibiting the 5αR activity, decreasing the DHT levels and in turn suppressing the expression of 5αR. Our study may contribute to the development of a new generation of herbal supplements with clearer material basis of pharmacodynamic for treating androgenic alopecia (AGA).

[Pharmacokinetic study of six aconitine alkaloids in aconiti lateralis radix praeparata in beagle dogs].

OBJECTIVE: To study the pharmacokinetics characteristics of six Aconitum alkaloids aconitine (AC), mesaconitine (MA), hypaconitine (HA), benzoylaconine (BAC), benzoylmesaconine (BMA) and benzoylhypaconine (BHA) in beagle dogs. METHODS: An ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed for simultaneous quantitation of six Aconitum alkaloids in beagle dog plasma after oral administration of Aconiti Lateralis Radix Praeparata decoction. UPLC/MS/MS system coupled with an electrospray ionization (ESI) source was performed in multiple-reaction monitoring (MRM) mode. Sample preparation was performed with solid-phase extraction(SPE) on a 3 mL HLB cartridge before the analysis. The separation was applied on a Waters C8 column (100 mm x 2.1 mm, 1.7 microm) and a gradient elution of methanol and 0.2% formic acid-water was used as mobile phase. The pharmacokinetic parameters were calculated by the results of the analysis through the DAS 2. 1 software (Drug and Statistics for Windows). RESULTS: The results showed that the fitting model for the six Aconitum alkaloids was the one-compartment model pharmacokinetics. CONCLUSION: The method is successfully used for the pharmacokinetic evaluation of the six Aconitum alkaloids in beagle dog plasma, it can help monitor the ADME/Tox process when taking Aconiti Lateralis Radix Praeparata by observing the pharmacokinetic process. The results provide a good reference for clinical treatment and safe application of Aconiti Lateralis Radix Praeparata.

[Study on effect of heavy metal cadmium ions on the ultramicrostructure damage of Pheretima aspergillum gastrointestinal epithelial cells].

OBJECTIVE: To study the effect of cadmium ions of different concentrations on gastrointestinal epithelial cells structure of Pheretima aspegillum (PA). METHODS: PA were contaminated with cadmium ions of different concentrations,and the structure of the body skin was observed, under light microscope and transmission electron microscope. RESULTS: With the increasing of cadmium ions concentration, a large number of lysosomal hyperplasia could be seen in the PA intestinal epithelial cells, the Golgi complex distributed around, and some Golgi complex hyperplasiaed, extended to a large bubble, microvilli, cilia arranged in irregular, disordered. While in the group contaminated with the high concentration cadmium ions, such as 30 mg/kg, the microvilli of the PA intestinal epithelial cells contracted, necrotic ulcer lesions occurred in the ciliated cells. CONCLUSION: The ultrastructure damage extent of PA gastrointestinal epithelial cells is dependent on the amount of the heavy metal contamination. PA with lower concentration Cd contamination shows mainly lysosomal proliferation, indicating heavy metal accumulation in lysosomes to eliminate toxic substances as a responsible reaction, this kind of damage is reversible. However, PA with higher concentration Cd contamination shows mainly microvilli and mitochondrial damage, nuclear membrane disintegration, nucleoplasm spillover, leading to necrosis, irreversible damage, indicating heavy metal accumulation of PA is related to this trait of intestinal epithelial cells.

[Study on the quantitative change of anthraquinonoids of Rhei in the preparation of dachengqi].

OBJECTIVE: To study the scientific evidence of the traditional preparation of Dachengqi: "Boiling Aurantii Immaturus and Magnoliae Officinalis first, and then adding Rhei to decoct together. Discarding the dregs, adding Natrii Sulfas into the decoction and drinking the upper solution when the Natrii Sulfas has dissolved completely". METHOD: The concentrations of free and combined anthraquinonoids(emodin, rhein, chrysophanol, physcion) in different decoctions were determined with HPLC method respectively. RESULT: When Natrii Sulfas, Aurantii Immaturus and Magnolias Officinalis are decocted with Rhei in different schemes, the concentrations of anthraquinonoids were changed regularly. CONCLUSION: The scientific evidence of traditional preparation method greatly increased the concentrations of the active components in Dachengqi.