3,4,5-tri-O-caffeoylquinic acid attenuates influenza A virus induced inflammation through Toll-like receptor 3/7 activated signaling pathway.

BACKGROUND: 3,4,5-tri-O-caffeoylquinic acid (3,4,5-TCQA), a natural polyphenolic acid, has been shown to be effective against influenza A virus (IAV) infection. Although it was found to inhibit the neuraminidase of IAV, it may also perturb other cellular functions, as polyphenolic acids have shown antioxidant, anti-inflammatory and other activities. PURPOSE: This study aimed to investigate the effect of 3,4,5-TCQA at a cell level, which is critical for protecting host cell from IAV infection. STUDY DESIGN AND METHODS: We explored the effect of 3,4,5-TCQA on H292 cells infected or un-infected with Pr8 IAV. The major genes and related pathway were identified through RNA sequencing. The pathway was confirmed by qRT-PCR and western blot analysis. The anti-inflammatory activity was evaluated using nitric oxide measurement assay. RESULTS: We showed that 3,4,5-TCQA downregulated the immune response in H292 cells, and reduced the cytokine production in Pr8-infected cells, through Toll-like receptor (TLR) signaling pathway. In addition, 3,4,5-TCQA showed anti-inflammatory activity in LPS-activated RAW264.7 cells. CONCLUSION: Collectively, our results indicated that 3,4,5-TCQA suppressed inflammation caused by IAV infection through TLR3/7 signaling pathway. This provides a new insight into the antiviral mechanism of 3,4,5-TCQA.

Characterization and phylogenetic analysis of the complete chloroplast genome of Curcuma comosa and C. latifolia.

Members of the Curcuma genus, a crop in the Zingiberaceae, are widely utilized rhizomatous herbs globally. There are two distinct species, C. comosa Roxb. and C. latifolia Roscoe, referred to the same vernacular name "Wan Chak Motluk" in Thai. C. comosa holds economic importance and is extensively used as a Thai traditional medicine due to its phytoestrogenic properties. However, its morphology closely resembles that of C. latifolia, which contains zederone, a compound known for its hepatotoxic effects. They are often confused, which may affect the quality, efficacy and safety of the derived herbal materials. Thus, DNA markers were developed for discriminating C. comosa from C. latifolia. This study focused on analyzing core DNA barcode regions, including rbcL, matK, psbA-trnH spacer and ITS2, of the authentic C. comosa and C. latifolia species. As a result, no variable nucleotides in core DNA barcode regions were observed. The complete chloroplast (cp) genome was introduced to differentiate between the two species. The comparison revealed that the cp genomes of C. comosa and C. latifolia were 162,272 and 162,289 bp, respectively, with a total of 133 identified genes. The phylogenetic analysis revealed that C. comosa and C. latifolia exhibited a very close relationship with other Curcuma species. The cp genome of C. comosa and C. latifolia were identified for the first time, providing valuable insights for species identification and evolutionary research within the Zingiberaceae family.

Ethnopharmacology of five flowers herbal tea, a popular traditional beverage in Hong Kong and South China.

BACKGROUND: It has been a long-standing tradition of using herbal tea for preventive and therapeutic healthcare in Hong Kong and South China and Five Flowers Tea is one of the most popular herbal teas. Based on the principle of traditional Chinese medicine, the pharmacological functions are to clear heat and dispel dampness in the body. Heat and dampness are thought to contribute to a range of health problems, especially during the hot and humid season in South China and Hong Kong. The most prevalent herbs in the formula contain bioactive compounds including flavonoids, alkaloids and terpenoids, which have a wide range of pharmacological properties including anti-inflammation, antivirus, antidiarrhoea, antibacteria, and antioxidation. However, with the composition varies widely, the ethnopharmacological benefits described may not be delivered uniformly. This study is to provide a comprehensive analysis on the composition of the Five Flowers Tea sold in Hong Kong and investigate the rationale behind the selection of herbs used in the formula. This study also provides information on the variation and quality of the Five Flowers Tea in the market. METHODS: Thirty-three Five Flowers Tea samples were collected from various locations in Hong Kong. The size, texture, colour and organoleptic properties were documented. Macroscopic and molecular authentication methods were employed to identify the individual components. RESULTS: Macroscopic identification revealed there were 23 herbs belonging to 18 plant families. The most prevalent herb was Bombax ceiba L., followed by Chrysanthemum morifolium. Ten adulterants and the existence of insect Lasioderma serricorne were confirmed by DNA barcoding techniques. CONCLUSION: This study employed a comprehensive approach to authenticate the herbs in Five Flowers Tea samples collected from various locations in Hong Kong. Macroscopic and molecular methods were used to identify the herbs and adulterants. The findings revealed the varied composition in Five Flowers Tea and the occurrence of adulterants in some samples. This shows that quality assurance of Five Flowers Tea is essential for the effective use of this popular folk medicine.

A systematic approach for authentication of medicinal Patrinia species using an integration of morphological, chemical and molecular methods.

Four common Patrinia species, including P. heterophylla, P. monandra, P. scabiosifolia and P. villosa, have been documented as herbal medicines with various clinical applications, such as anti-cancer, anti-diarrhea and sedative. However, the authentication of medicinal Patrinia species poses a problem, particularly with the processed herbal materials. This study aimed to systematically authenticate the four medicinal Patrinia species in the market using morphological and chemical characterization, as well as DNA markers. We found the species identity authenticated by traditional morphologies were in good agreement with both chemical and molecular results. The four species showed species-specific patterns in chromatographic profiles with distinct chemical markers. We also revealed the power of complete chloroplast genomes in species authentication. The sequences of targeted loci, namely atpB, petA, rpl2-rpl23 and psaI-ycf4, contained informative nucleotides for the species differentiation. Our results also facilitate authentication of medicinal Patrinia species using new DNA barcoding markers. To the best of our knowledge, this is the first report on the application of morphology, chemical fingerprinting, complete chloroplast genomes and species-specific Insertion-Deletions (InDels) in differentiating Patrinia species. This study reported on the power of a systematic, multidisciplinary approach in authenticating medicinal Patrinia species.

Determination of ITS1 haplotypes of Fritillariae Cirrhosae Bulbus by amplicon sequencing.

BACKGROUND: Fritillariae Cirrhosae Bulbus is an antitussive and expectorant Chinese medicinal material derived from the dried bulbs of six Fritillaria species. In the 2015 edition of the Chinese Pharmacopoeia, the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) is the officially listed method for their authenfication. Specifically, the ~ 300-bp ITS1 amplicon of only Fritillariae Cirrhosae Bulbus but not other Fritillaria species can be cleaved into two smaller fragments with restriction enzyme SmaI. Considering repeated reported cases of incomplete digestion of ITS1 amplicon, this study aims to investigate the possibility of heterogeneous ITS1 sequences contained in the Fritillariae Cirrhosae Bulbus. METHODS: In this study, ITS1 amplicons of Fritillaria Cirrhosae Bulbus and four other Fritillaria species were sequenced on Illumina platform. We utilised high-throughout amplicon sequencing to determine ITS1 haplotypes and their frequencies in Fritillaria genomes. RESULTS: Our results showed that all six botanical sources of Fritillariae Cirrhosae Bulbus indeed possess ITS1 haplotypes with no SmaI restriction site, and the average percentages of ITS1 reads containing SmaI restriction site ranged from 63.60% to 91.81%. CONCLUSION: Our findings suggest that the incomplete digestion in PCR-RFLP analysis of Fritillariae Cirrhosae Bulbus is caused by the presence of ITS1 haplotypes without SmaI restriction site due to intragenomic heterogeneity.

Anti-influenza virus activities and mechanism of antrafenine analogs.

Antrafenine is a drug initially designed for anti-inflammation uses. In this work we have synthesized a library of its structural analogs and tested the anti-influenza activities. These analogs belong to a group of 2-(quinolin-4-yl)amino benzamides or 2-(quinolin-4-yl)amino benzoate derivatives. Best performers were identified, namely 12, 34, 41, with IC50 against A/WSN/33 (H1N1) of 5.53, 3.21 and 6.73 µM respectively. These chemicals were also effective against A/PR/8/34 (H1N1), A/HK/1/68 (H3N2) and B/Florida/04/2006 viruses. Time-of-addition study and minigenome luciferase reporter assay both supported that the compounds act on the ribonucleoprotein (RNP) components. Using 34 and 41 as representative compounds, we determined by microscale thermophoresis that this group of compounds bind to both PA C-terminal domain and the nucleoprotein (NP) which is the most abundant subunit of the RNP. Taken together, we have identified a new class of anti-influenza compounds with dual molecular targets and good potential to be further developed. IMPORTANCE: The influenza viruses, especially influenza A and B subtypes, cause many deaths each year. The high mutation rate of the virus renders available therapeutics less effective with time. In this work we identify a new class of compounds, structurally similar to the anti-inflammation drug antrafenine, with good potency against influenza A strains. The IC50 of the best performers are within low micromolar range and thus have good potential for further development.

DNA from herbs can be obtained from air and authenticated by polymerase chain reaction.

DNA barcoding of herbs allows accurate species authentication. However, the DNA of herbs are often not easily PCR amplified due to co-extraction of inhibitors. Methods have been developed to improve DNA extraction to reduce contaminants. These methods usually require toxic chemical treatments or expensive commercial kits and are labor intensive. In this report, we collected the air passed from the herbs and directly amplified the DNA obtained. Results showed that DNA could be obtained, and it was PCR amplifiable. Sequencing of the amplified DNA allowed species authentication. This DNA collection method is applicable to herbs from different plant tissues. It has the advantages of reducing the use of toxic substances and more economical.

Berberine Reduces Lipid Accumulation in Obesity via Mediating Transcriptional Function of PPARδ.

Obesity is defined as a dampness-heat syndrome in traditional Chinese medicine. Coptidis Rhizoma is an herb used to clear heat and eliminate dampness in obesity and its complications. Berberine (BBR), the main active compound in Coptidis Rhizoma, shows anti-obesity effects. Peroxisome proliferator-activated receptors (PPARs) are a group of nuclear receptor proteins that regulate the expression of genes involved in energy metabolism, lipid metabolism, inflammation, and adipogenesis. However, whether PPARs are involved in the anti-obesity effect of BBR remains unclear. As such, the aim of this study was to elucidate the role of PPARs in BBR treatment on obesity and the underlying molecular mechanisms. Our data showed that BBR produced a dose-dependent regulation of the levels of PPARγ and PPARδ but not PPARα. The results of gene silencing and specific antagonist treatment demonstrated that PPARδ is key to the effect of BBR. In 3T3L1 preadipocytes, BBR reduced lipid accumulation; in high-fat-diet (HFD)-induced obese mice, BBR reduced weight gain and white adipose tissue mass and corrected the disturbed biochemical parameters, including lipid levels and inflammatory and oxidative markers. Both the in vitro and in vivo efficacies of BBR were reversed by the presence of a specific antagonist of PPARδ. The results of a mechanistic study revealed that BBR could activate PPARδ in both 3T3L1 cells and HFD mice, as evidenced by the significant upregulation of PPARδ endogenous downstream genes. After activating by BBR, the transcriptional functions of PPARδ were invoked, exhibiting negative regulation of CCAAT/enhancer-binding protein α (Cebpα) and Pparγ promoters and positive mediation of heme oxygenase-1 (Ho-1) promoter. In summary, this is the first report of a novel anti-obesity mechanism of BBR, which was achieved through the PPARδ-dependent reduction in lipid accumulation.

Berberine activates PPARδ and promotes gut microbiota-derived butyric acid to suppress hepatocellular carcinoma.

BACKGROUND: Peroxisome proliferator-activated receptors (PPARs) are a family of ligand-inducible transcription factors that govern various essential metabolic activities in the liver and other organs. Recently, berberine (BBR) has been characterized as a modulator of PPARs; however, the matter of whether PPARs are involved in the inhibitory effect of BBR on hepatocellular carcinoma (HCC) is not well understood. PURPOSE: This study aimed to investigate the role of PPARs in the suppressive effect of BBR on HCC and to elucidate the relative mechanism. METHODS: We studied the role of PPARs in the anti-HCC effects of BBR both in vitro and in vivo. The mechanism whereby BBR regulated PPARs was studied using real-time PCR, immunoblotting, immunostaining, luciferase, and a chromatin immunoprecipitation coupled PCR assay. Additionally, we used adeno-associated virus (AAV)-mediated gene knockdown to address the effect of BBR more effectively. RESULTS: We demonstrated that PPARδ played an active role in the anti-HCC effect of BBR, rather than PPARα or PPARγ. Following a PPARδ-dependent manner, BBR increased BAX, cleaved Caspase 3, and decreased BCL2 expression to trigger apoptotic death, thereby suppressing HCC development both in vitro and in vivo. It was noted that the interactions between PPARδ and the apoptotic pathway resulted from the BBR-induced upregulation of the PPARδ transcriptional function; that is, the BBR-induced activation of PPARδ could mediate the binding with the promoters of apoptotic genes such as Caspase 3, BAX, and BCL2. Moreover, gut microbiota also contributed to the suppressive effect of BBR on HCC. We found that BBR treatment restored the dysregulated gut microbiota induced by the liver tumor burden, and a functional gut microbial metabolite, butyric acid (BA), acted as a messenger in the gut microbiota-liver axis. Unlike BBR, the effects of BA suppressing HCC and activating PPARδ were not potent. However, BA was able to enhance the efficacy of BBR by reducing PPARδ degradation through a mechanism to inhibit the proteasome ubiquitin system. Additionally, we found that the anti-HCC effect of BBR or a combination of BBR and BA was much weaker in mice with AAV-mediated PPARδ knockdown than those in the control mice, suggesting the critical role of PPARδ. CONCLUSION: In summary, this study is the first to report that a liver-gut microbiota-PPARδ trilogy contributes to the anti-HCC effect of BBR. BBR not only directly activated PPARδ to trigger apoptotic death but also promoted gut microbiota-derived BA production, which could reduce PPARδ degradation to enhance the efficacy of BBR.

The Complete Chloroplast Genomes of Nine Smilacaceae Species from Hong Kong: Inferring Infra- and Inter-Familial Phylogeny.

The Smilacaceae is a cosmopolitan family consisting of 200-370 described species. The family includes two widely accepted genera, namely Smilax and Heterosmilax. Among them, the taxonomical status of Heterosmilax has been continuously challenged. Seven Smilax and two Heterosmilax species can be found in Hong Kong, with most of them having medicinal importance. This study aims to revisit the infra-familial and inter-familial relationships of the Smilacaceae using complete chloroplast genomes. The chloroplast genomes of the nine Smilacaceae species from Hong Kong were assembled and annotated, which had sizes of 157,885 bp to 159,007 bp; each of them was identically annotated for 132 genes, including 86 protein-coding genes, 38 transfer RNA genes, and 8 ribosomal RNA genes. The generic status of Heterosmilax was not supported because it was nested within the Smilax clade in the phylogenetic trees, echoing previous molecular and morphological studies. We suggest delimitating the genus Heterosmilax as a section under the genus Smilax. The results of phylogenomic analysis support the monophyly of Smilacaceae and the exclusion of Ripogonum from the family. This study contributes to the systematics and taxonomy of monocotyledons, authentication of medicinal Smilacaceae, and conservation of plant diversity.

Bioassay guided isolation of caffeoylquinic acids from the leaves of Ilex pubescens Hook. et Arn. and investigation of their anti-influenza mechanism.

ETHNOPHARMACOLOGICAL RELEVANCE: Ilex pubescens Hook. et Arn. (Maodongqing, MDQ) is a common herbal tea ingredient in Southern China for heat clearance and anti-inflammation. Our preliminary screening showed that 50% ethanol extract of its leaves has anti-influenza virus activity. In this report, we proceed to identify the active components and clarify the related anti-influenza mechanisms. AIM: We aim to isolate and identify the anti-influenza virus phytochemicals from the extract of the MDQ leaves, and study their anti-influenza virus mechanism. MATERIAL AND METHODS: Plaque reduction assay was used to test the anti-influenza virus activity of fractions and compounds. Neuraminidase inhibitory assay was used to confirm the target protein. Molecular docking and reverse genetics were used to confirm the acting site of caffeoylquinic acids (CQAs) on viral neuraminidase. RESULTS: Eight CQAs, 3,5-di-O-caffeoylquinic acid methyl ester (Me 3,5-DCQA), 3,4-di-O-caffeoylquinic acid methyl ester (Me 3,4-DCQA), 3,4,5-tri-O-caffeoylquinic acid methyl ester (Me 3,4,5-TCQA), 3,4,5-tri-O-caffeoylquinic acid (3,4,5-TCQA), 4,5-di-O-caffeoylquinic acid (4,5-DCQA), 3,5-di-O-caffeoylquinic acid (3,5-DCQA), 3,4-di-O-caffeoylquinic acid (3,4-DCQA), and 3,5-di-O-caffeoyl-epi-quinic acid (3,5-epi-DCQA) were identified from the MDQ leaves, in which Me 3,5-DCQA, 3,4,5-TCQA and 3,5-epi-DCQA were isolated for the first time. All these eight compounds were found to inhibit neuraminidase (NA) of influenza A virus. The results of molecular docking and reverse genetics indicated that 3,4,5-TCQA interacted with Tyr100, Gln412 and Arg419 of influenza NA, and a novel NA binding groove was found. CONCLUSION: Eight CQAs isolated from the leaves of MDQ were found to inhibit influenza A virus. 3,4,5-TCQA was found to interact with Tyr100, Gln412 and Arg419 of influenza NA. This study provided scientific evidence on the use of MDQ for treating influenza virus infection, and laid the foundation for the development of CQA derivatives as potential antiviral agents.

Functional Importance of the Hydrophobic Residue 362 in Influenza A PB1 Subunit.

PB1, acting as the catalytic subunit of the influenza polymerase, has numerous sequentially and structurally conserved regions. It has been observed that the slight modification of residues in PB1 would greatly affect the polymerase activity and even host adaptation ability. Here, we identified a critical residue, 362M, on the polymerase activity and virus replication. By means of the minireplicon assay, we assured the importance of the hydrophobicity of PB1 362, and the possibility that the size and charge of the side chain might directly interfere with the polymerase function. We also proposed a hydrophobic core between the PA-arch and the PB1 ß-hairpin motifs and showed the importance of the core to the polymerase function.

Characterization and phylogenetic analysis of the complete chloroplast genome of Emilia sonchifolia (L.) DC.

Emilia sonchifolia is a herb with antioxidant, anti-inflammatory, antitumor, and wound healing properties. The complete chloroplast genome (cp genome) of the genus Emilia was sequenced for the first time. The cp genome of E. sonchifolia is 151,474 bp in length. It contained a large single-copy (LSC) region (84,004 bp), and small single-copy (SSC) region (17,980 bp), and two inverted repeats (IRs, 24,745 bp). Phylogenetic analysis of 24 species was conducted. E. sonchifolia was found to be closely related to Pericallis hybrida and Dendrosenecio spp. The sequenced cp genome would be useful to understand the phylogeny and genomic studies of the genus Emilia.

Comparison of DNA extraction methods on CITES-listed timber species and application in species authentication of commercial products using DNA barcoding.

Quality and quantity of DNA extracted from wood is important for molecular identification of wood species, which can serve for conservation of wood species and law enforcement to combat illegal wood trading. Rosewood (Dalbergia and Pterocarpus) and agarwood (Aquilaria) are the most commonly found hardwood in timber seizure incidents. To monitor international trade of timber and commercial wood products and to protect these endangered wood species from further population decline, in this study, we have compared three extraction protocols for DNA extraction from 12 samples of rosewood and agarwood timber logs, and later applied the best DNA extraction protocol on 10 commercial wood products claimed to be rosewood and agarwood. We also demonstrated the applicability of DNA mini-barcoding with multi-loci combination with reference library for identifying the species of timber and commercial wood products. We found that a silica column-based method with guanidine thiocyanate-containing binding buffer served the best in DNA extraction from different parts of wood in all three genera with good quality and quantity. Single barcode region ITS2 or multi-loci combinations including ITS2 barcode region generally provide better discriminatory power for species identification for both rosewood and agarwood. All 10 products were identified to species-level using multi-loci combination. In terms of accuracy in labelling, 80% of them were labelled correctly. Our work has shown the feasibility of extracting good quality of DNA from authentic wood samples and processed wood products and identifying them to species level based on DNA barcoding technology.

Glycosylation of the polyphenols from Resina draconis by glycosyltransferase YjiC1.

Resina Draconis (RD), also known as 'dragon's blood', contains a broad range of natural compounds, such as flavonoids, stilbenes and dihydrochalcones. It is clinically used to enhance blood circulation. However, the major components of RD suffer from relatively poor water solubility. Glycosylation is a critical determinant for modulating solubility and improving bioavailability and bioactivity of natural products. Herein, we report a novel method to efficiently synthesize glycosidic derivatives of the major polyphenols in RD using a microbial glycosyltransferase, i.e., YjiC1. Solubility test showed that the synthetic glycosidic derivatives displayed higher water solubility than the raw materials. This research sheds light on the structural modification of natural products for higher water solubility, which is important for innovative drug discovery.

Anti-influenza properties of tiliroside isolated from Hibiscus mutabilis L.

ETHNOPHARMACOLOGICAL RELEVANCE: Fu Rong Ye (FRY), the leaf of Hibiscus mutabilis L., is a Chinese medicinal herb used to treat coughs and respiratory diseases. FRY is the major herbal component of the patent medicine Fupo Ganmao Granules for treating common cold. However, its anti-influenza active components and mechanism were not identified. AIM: Here, we aim to a) isolate the anti-influenza phytochemicals from FRY extract and b) explore its anti-flu mechanism. MATERIAL AND METHODS: Bioassay guided isolation was performed to get anti-influenza virus components. Influenza virus infected cells and mouse model were employed for efficacy evaluation. RESULTS: Using bioassay-guided isolation, the flavonoid tiliroside was obtained, which inhibited four IAV strains in MDCK cells with EC50 ranging from 3.87 to 27.61 µM by suppressing the viral ribonucleoprotein activity. Tiliroside also significantly downregulated the expression of cytokines/chemokines in A549 cells, and protected 50% of PR8-infected BALB/c mice from death and at 800 mg/kg/day, improved lung edema conditions. CONCLUSION: Tiliroside is effective for influenza virus infection treatment and promising for further drug development. This study is the first to demonstrate that tiliroside in FRY acts against influenza virus.

Differentiation of Lingxiaohua and Yangjinhua by chloroplast genome sequencing and DNA barcoding markers.

Lingxiaohua (Campsis Flos, Campsis grandiflora (Thunb.) K. Schum) is a medicinal herb used for promoting diuresis and treating blood-related disorders by the promotion of blood circulation. It also possesses anti-inflammatory and antioxidative properties. This non-poisonous plant is frequently confused with poisonous Yangjinhua (Daturae Metelis Flos, Datura metel Linnaeus) in the market, resulting in serious anticholinergic poisoning. The confusion of these two herbs is due to the similarity in their appearances. In our study, we compared the complete chloroplast genomes of the two plants and found that they are very different in terms of their gene content and gene arrangement. There were also significant differences in the number and repeating motifs of microsatellites and complex repeats. We used universal primers for the amplification of rbcL, matK, psbA-trnH, and ITS2 regions and successfully differentiated the two plants. Furthermore, we designed two pairs of primers based on the nucleotide differences in chloroplast genomes at the rps14 and rpoC1 regions to provide additional authentication markers. The universal primers and specific primers when used together can accurately discriminate Lingxiaohua and Yangjinhua.

Interaction of Influenza A Nucleoprotein with Host hnRNP-C Is Implicated in Viral Replication.

The host interactome of influenza viral proteins is ever-expanding. In this work, we report the identification of host heterogeneous nuclear ribonucleoprotein C (hnRNP-C) as an interacting partner of influenza A virus nucleoprotein (NP). We confirmed that this interaction exists across different influenza A subtypes and strains. Using biochemical methods, we determined that hnRNP-C interacts with NP via its C-terminal auxiliary domain. Further, we determined that the hnRNP-C is a negative regulator of influenza viral growth. Its interaction with NP is implicated in the promotion of host cell apoptosis during viral infection. It is the first time that the interaction between influenza nucleoprotein and host heterogeneous nuclear ribonucleoprotein C is characterized in detail. Overall, these findings not only characterize the interaction between NP and its host interacting partner hnRNP-C but also clarify the functional significance of this interaction. This work may lead to a new therapeutic target for the development of anti-influenza drugs.

Urolithin M5 from the Leaves of Canarium album (Lour.) DC. Inhibits Influenza Virus by Targeting Neuraminidase.

Ganlanye (GLY), the leaf of Canarium album (Lour.) DC., is a traditional Chinese medicinal herb for warm disease treatment. We found that its aqueous extract could inhibit the influenza A virus. To find and characterize anti-influenza virus phytochemicals from GLY, we performed (1) bioassay-guided isolation, (2) a cell and animal assay, and (3) a mechanism study. Bioassay-guided isolation was used to identify the effective components. Influenza virus-infected MDCK cell and BALB/c mouse models were employed to evaluate the anti-influenza virus activities. A MUNANA assay was performed to find the NA inhibitory effect. As a result, urolithin M5 was obtained from the crude extract of GLY. It inhibited influenza virus activities in vitro and in vivo by suppressing the viral NA activity. In the MDCK cell model, urolithin M5 could inhibit an oseltamivir-resistant strain. In a PR8-infected mouse model, 200 mg/kg/d urolithin M5 protected 50% of mice from death and improved lung edema conditions. GLY was recorded as a major traditional herb for warm disease treatment. Our study identified GLY as a potent anti-influenza herb and showed urolithin M5 as the active component. We first report the in vivo activity of urolithin M5 and support the anti-influenza application of GLY.

Molecular Identification of Commercial Fish Maws by DNA Sequencing of 16S rRNA and Cytochrome c Oxidase I Genes.

ABSTRACT: Fish maws (dried swim bladders) have long been used for medicinal tonics and as a valuable food resource in Southeast Asia. However, it is difficult to identify the original species of fish maws sold in markets due to a lack of taxonomic characteristics. In the present study, 37 kinds of commercial fish maws from various medicinal material markets were examined, and gene sequences were successfully obtained from ca. 95% of the samples. Partial sequences of the 16S rRNA gene and cytochrome c oxidase I (COI) gene were obtained and used to investigate the origin of these commercial fish maws. Thirty-five specimens belonged to nine species: five croakers and four noncroakers. All species identification was supported by both high homogeneity (98 to 100%) and clear clustering with low within-group Kimura two-parameter divergence scores (0 to 0.04 for 16S rRNA and 0 to 0.07 for COI) and high between-group divergence scores (0.07 to 0.15 for 16S rRNA and 0.11 to 0.24 for COI). Croakers were the predominant species, accounting for 74% of the total fish maw specimens. The large demand for croakers has put some species at the risk of extinction due to overfishing. As a valuable food, fish maw has progressively become more popular and has been used as a substitute for shark fin. The identification results allowed us to learn more about the fish species available on the fish maw market and provided an indicator for possible control of threatened or endangered fish species. A probable correlation between the molecular characteristics and morphological features of fish maws was also found and could provide both consumers and merchants with an important reference for identifying the origin of fish maws.