Anoectochilus roxburghii polysaccharide reduces D-GalN/LPS-induced acute liver injury by regulating the activation of multiple inflammasomes.

BACKGROUND: Acute liver injury (ALI) is a serious syndrome with a high mortality rate due to viral infection, toxic exposure, and autoimmunity, and its severity can range from mildly elevated liver enzymes to severe liver failure. Activation of the nod-like receptor pyrin domain-containing 3 (NLRP3) inflammasome is closely associated with the development of ALI, and the search for an inhibitor targeting this pathway may be a novel therapeutic option. Anoectochilus roxburghii polysaccharide (ARP) is a biologically active ingredient extracted from Anoectochilus roxburghii with immunomodulatory, antioxidant, and anti-inflammatory bioactivities and pharmacological effects. In this study, we focused on D-galactosamine (D-GalN)/lipopolysaccharide (LPS)-induced acute liver injury by ARP through inhibition of NLRP3 inflammasome. METHODS: An inflammasome activation model was established in bone marrow-derived macrophages (BMDMs) to investigate the effects of ARP on caspase-1 cleavage, IL-1ß secretion, and ASC oligomerization in inflammasomes under different agonists. We used the D-GalN/LPS-induced acute liver injury model in mice, intraperitoneally injected ARP or MCC950, and collected liver tissues, serum, and intraperitoneal lavage fluid for pathological and biochemical indexes. RESULTS: ARP effectively inhibited the activation of the NLRP3 inflammasome and had an inhibitory effect on non-classical NLRP3, AIM2, and NLRC4 inflammasomes. It also effectively inhibited the oligomerization of apoptosis-associated speck-like protein (ASC) from a variety of inflammatory vesicles. Meanwhile, ARP has good therapeutic effects on acute liver injury induced by D-GaIN/LPS. CONCLUSION: The inhibitory effect of ARP on a wide range of inflammasomes, as well as its excellent protection against acute liver injury, suggests that ARP may be a candidate for acute liver injury.

Physiological and transcriptomic analyses reveal the regulatory mechanisms of Anoectochilus roxburghii in response to high-temperature stress.

BACKGROUND: High temperatures significantly affect the growth, development, and yield of plants. Anoectochilus roxburghii prefers a cool and humid environment, intolerant of high temperatures. It is necessary to enhance the heat tolerance of A. roxburghii and breed heat-tolerant varieties. Therefore, we studied the physiological indexes and transcriptome of A. roxburghii under different times of high-temperature stress treatments. RESULTS: Under high-temperature stress, proline (Pro), H2O2 content increased, then decreased, then increased again, catalase (CAT) activity increased continuously, peroxidase (POD) activity decreased rapidly, then increased, then decreased again, superoxide dismutase (SOD) activity, malondialdehyde (MDA), and soluble sugars (SS) content all decreased, then increased, and chlorophyll and soluble proteins (SP) content increased, then decreased. Transcriptomic investigation indicated that a total of 2740 DEGs were identified and numerous DEGs were notably enriched for "Plant-pathogen interaction" and "Plant hormone signal transduction". We identified a total of 32 genes in these two pathways that may be the key genes for resistance to high-temperature stress in A. roxburghii. CONCLUSIONS: To sum up, the results of this study provide a reference for the molecular regulation of A. roxburghii's tolerance to high temperatures, which is useful for further cultivation of high-temperature-tolerant A. roxburghii varieties.

Phytochemical Research and Anti-inflammatory Activities of the Ethanol Extract from Anoectochilus roxburghii (Wall.) Lindl.

Anoectochilus roxburghii is a well-known and valuable traditional Chinese herb due to various medicinal and functional benefits. In-depth investigation is necessary to discover active ingredients and expand its application. In this study, four new compounds (1-4) along with ten known compounds (5-14) were isolated from the ethanol extract ofA.roxburghii. Their structures were elucidated by spectroscopic data interpretation. The isolates were screened for their inhibitory activities on the production of NO in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. Among them, compounds 5, 6, 9, 10, 12, 13 and 14exhibited significant anti-inflammatory activity through inhibiting the release of NO.

Effects of compound Anoectochilus roxburghii (Wall.) Lindl. oral liquid on relative metabolic enzymes and various biochemical indices in Wistar rats with isoniazid-induced liver injury.

Isoniazid (INH) is the first-line anti-tuberculosis drug in clinical practice, and its main adverse effect is drug-induced liver injury (DILI). This study aimed to investigate the hepatoprotective effect of Compound Anoectochilus roxburghii (Wall.) Lindl. Oral Liquid (CAROL) and to provide a new strategy for the search of potential drugs against INH-induced liver injury in Wistar rats. Animal experiment was based on INH (100 mg/kg) induced liver injury to explore the intervention effects of CAROL at doses of 1.35, 2.70, and 5.40 mL/kg. LC-QTOF-MS/MS was used to identify hepatoprotective components in CAROL and its' exposed components in rat serum. The hepatoprotective effect of CAROL was evaluated by pathological observation of rat liver tissue and changes in levels of biochemical indices and cytokines in serum or liver tissue. Of the 58 hepatoprotective components identified, 15 were detected in the serum of rats with liver-injured treated by high-dose CAROL. Results of animal experiments showed that the levels of various biochemical indexes and cytokines were significantly reversed with CAROL intervention. In particular, the expression level of cytokeratin-18 and high-mobility group box 1, as specific and sensitive indicators of DILI, was significantly reduced in the serum of rats with CAROL intervention compared with the INH model group. The same reversal was observed in the levels of TBIL, ALP, ALT, and AST in serum, as well as in the levels of TNF-α, IL-6, SOD, and MDA in liver tissue. For INH-metabolizing enzymes, an evident expression inhibition was observed in N-acetyltransferase 2 and glutathione S-transferases with CAROL intervention, which may be the key to controlling INH hepatotoxicity. CAROL has a favorable hepatoprotective effect on INH-induced liver injury. This study takes the first step in studying the hepatoprotective mechanism of CAROL against INH hepatotoxicity and provides reference for wider clinical applications.

Gooderoside A from Anoectochilus elatus attenuates acute and chronic pains by inhibiting NO/cGMP and IRAK4/IRAK1/TAK1 signaling pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: Anoectochilus elatus Lindl. was traditionally used for pain treatment and Gooderoside A (GA) was regarded as its principal constituent. AIM OF THE STUDY: To investigate whether GA can be responsible for the antinociceptive activity of A. elatus and explore its underlying mechanism. MATERIALS AND METHODS: Acetic acid-induced abdominal writhing and tail flick tests were employed to evaluate the antinociceptive activity of ethanolic extract of A. elatus (EEA) and GA. Formalin test was used to ascertain the antinociceptive pattern of GA. Entobarbital sodium induced sleep test was adopted to exclude its hypnotic effect, while open-field test was performed to rule out its motor impairment effect. Chronic constriction injury (CCI)-induced neuropathic pain in rats was developed to evaluate its efficacy on neuropathic pain, and BV-2 cells were used to explore the underlying mechanism. RESULTS: EEA and GA, significantly inhibited chemical and thermal nociception. GA suppressed nociception in formalin test in both phase I and II, whereas methylene blue and L-NAME partially reversed its efficacy. GA located inner and slightly blocked sodium channel current, and did not show any hypnotic effect or motor impairment effect. Crucially, GA markedly attenuated chronic neuropathic pain in rats, inhibited the phosphorylation of IRAK4, IRAK1 and TAK1, and suppressed MAPKs pathway in BV-2 cells. CONCLUSION: GA relieved acute and chronic pains in vivo. The mechanism of action involves the blocking of NO/cGMP and IRAK4/IRAK1/TAK1 pathways. These results suggested GA may be a promising candidate for antinociceptive drug development.

Biochemical and Transcriptome Analysis Reveals Pigment Biosynthesis Influenced Chlorina Leaf Formation in Anoectochilus roxburghii (Wall.) Lindl.

Anoectochilus roxburghii (Wall.) Lindl is a perennial herb of the Orchidaceae family; a yellow-green mutant and a yellow mutant were obtained from the wild type, thereby providing good material for the study of leaf color variation. Pigment content analysis revealed that chlorophyll, carotenoids, and anthocyanin were lower in the yellow-green and yellow mutants than in the wild type. Transcriptome analysis of the yellow mutant and wild type revealed that 78,712 unigenes were obtained, and 599 differentially expressed genes (120 upregulated and 479 downregulated) were identified. Using the Kyoto Encyclopedia of Genes and Genomes pathway analysis, candidate genes involved in the anthocyanin biosynthetic pathway (five unigenes) and the chlorophyll metabolic pathway (two unigenes) were identified. Meanwhile, the low expression of the chlorophyll and anthocyanin biosynthetic genes resulted in the absence of chlorophylls and anthocyanins in the yellow mutant. This study provides a basis for similar research in other closely related species.

Anti-COVID-19 mechanism of Anoectochilus roxburghii liquid based on network pharmacology and molecular docking / 药学学报

italic>Anoectochilus roxburghii liquid (spray, a hospital preparation of Wu Mengchao Hepatobiliary Hospital of Fujian Medical University) has shown a good clinical treatment effect during the COVID-19 pandemic, but its material basis and mechanism of action are still unclear. In this study, network pharmacology and molecular docking methods were used to predict the molecular mechanism of A. roxburghii liquid against COVID-19, and pharmacodynamic experiments in vitro were conducted to study the interaction between the current targets with clear preventive and therapeutic effects and the key components of A. roxburghii liquid. UPLC-MS and database were used to compare and analyze the active ingredients in the liquid, and 17 potential active ingredients with good drug-like properties were screened by in vivo pharmacokinetics process in SwissADME database. SwissTargetPrediction and GeneCards were searched to find 93 common targets. Cytoscape 3.8.2 software was used to construct the "component-target" network map, and the Metascape platform was used for gene function annotation and pathway enrichment analysis. It was found that the extract could regulate the positive response to external stimuli, inflammatory response, cytokine production and other biological processes by binding the active ingredients such as isorhamnetin, kaempferol, luteolin, quercetin and apigenin to the common targets (NOS3, MPO, MMP3, etc.), and play an anti-COVID-19 role. In the angiotensin-converting enzyme 2 (ACE2) activity inhibition assay, it was found that the stock solution of A. roxburghii liquid (for spray), and the supernatant after removing polysaccharides (mainly containing flavonoids) could to some extent inhibit the activity of ACE2. Crucially, in the experiment of 2019-nCOV-S pseudovirus infecting HEK-293T-ACE2 cells, we found that A. roxburghii liquid may exert anti-COVID-19 effects by blocking the binding of SARS-CoV-S protein to ACE2.

The evaluation of the anti-cancer effects of Anoectochilus roxburghii on hematological cancers in vitro.

The cause of hematological cancers is the uncontrolled proliferation of hematopoietic and lymphoid tissues, and chemotherapy is used to treat cancer. However, adverse side effects of chemotherapy are common. Therefore, the use of plant extracts as a method for treating cancer is becoming increasingly popular. Anoectochilus roxburghii (wall.) Lindl. (A. roxburghii) is one of the original sources of the valuable medicinal plants known as the king medicine and the golden grass. This study investigated the potential anticancer effect of A. roxburghi (AR) on JURKAT, MM1S, THP1 and U266 cells. To test the cytotoxic and apoptotic effects of AR, hematological cancer cells were exposed to increasing doses of AR (0.1-0.5 µg/µl). The spectrophotometric MTT assay and the flow cytometric Annexin V staining were used to examine the viability and apoptosis of the cells, respectively. qRT-PCR was used to determine the expression levels of the apoptosis-related genes BAD, BAX, BIM and BCL-2. Our results show that AR treatment decreased cell viability and induced apoptosis in each cell line. Our RT-PCR data showed that AR significantly increased the expression levels of the pro-apoptotic BAX gene in JURKAT and MM1S cells, whereas it significantly increased the expression levels of both BAX and BIM in U266 cells. This is the first study to investigate how AR modulates apoptosis in hematological cancer cells. As a result, AR therapy may be a promising treatment modality for the treatment of cancer.

Anoectochiluszhongshanensis (Orchidaceae), a new species from Guangxi, China.

A new species of Anoectochilus (Orchidaceae) from Guangxi, China, A.zhongshanensis, is described here, which was identified based on phylogenetic studies adopting combined plastid markers (rbcL-matK-trnL-F), morphological observation and chemical analysis. Molecular phylogenetic results support the systematic status of A.zhongshanensis as a new species in Anoectochilus genus. Morphologically, this new species is similar to A.zhejiangensis and A.malipoensis, but differs by its characteristic labellum and column, including the hastate or scalpel-shaped lobes of epichile, forward curved and pinnately divided cristate lobes at both sides of the mesochile and inverted triangle column wings. Furthermore, HPLC-ELSD analysis of these three species revealed the interesting chemotaxonomic difference that the principle and characteristic lactone glycoside in this new species was kinsenoside, rather than its diastereoisomer, goodyeroside A, a major glycoside in A.zhejiangensis and A.malipoensis.

Mechanism of the Effect of Compound Anoectochilus roxburghii (Wall.) Lindl. Oral Liquid in Treating Alcoholic Rat Liver Injury by Metabolomics.

Purpose: Compound Anoectochilus roxburghii (Wall.) Lindl oral liquid (CAROL) is often as a hepatoprotective agent. The present study aimed to elucidate the protective mechanism of CAROL against alcoholic liver injury in rats by untargeted metabolomics combined with multivariate statistical analysis. Methods: An alcoholic liver disease model was established in sprague-dawley (SD) rats by gavage of alcohol, and CAROL treatment was administered. The hepatoprotective effect of CAROL was evaluated by examining liver tissues changes and detecting biochemical index activities and cytokines in serum and liver homogenates. The metabolites in serum samples were examined using ultrahigh-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UHPLC-QTOF/MS) and multivariate statistical analysis to screen for differentially expressed metabolites and Kyoto Encyclopedia of Genes and Genomes (KEGG) to assess potential metabolic pathways. Results: CAROL has the potential to downregulate inflammation levels and alleviate oxidative stress. The differential metabolites are mainly engaged in riboflavin metabolism, arginine and proline metabolism, phenylalanine, tyrosine and tryptophan biosynthesis metabolism, phenylalanine metabolism, pyrimidine metabolism, and vitamin B6 metabolism to achieve hepatoprotective effects. Conclusion: CAROL may exhibit beneficial hepatoprotective effects by reducing inflammation, mitigating oxidative stress, and modulating metabolites and their metabolic pathways.This study has important implications for advancing the clinical application of CAROL.

The Toxicological Assessment of Anoectochilus burmannicus Ethanolic-Extract-Synthesized Selenium Nanoparticles Using Cell Culture, Bacteria, and Drosophila melanogaster as Suitable Models.

Selenium nanoparticles (SeNPs) are worthy of attention and development for nutritional supplementation due to their health benefits in both animals and humans with low toxicity, improved bioavailability, and controlled release, being greater than the Se inorganic and organic forms. Our previous study reported that Anoectochilus burmannicus extract (ABE)-synthesized SeNPs (ABE-SeNPs) exerted antioxidant and anti-inflammatory activities. Furthermore, ABE could stabilize and preserve the biological activities of SeNPs. To promote the ABE-SeNPs as supplementary and functional foods, it was necessary to carry out a safety assessment. Cytotoxicity testing showed that SeNPs and ABE-SeNPs were harmless with no killing effect on Caco2 (intestinal epithelial cells), MRC-5 (lung fibroblasts), HEK293 (kidney cells), LX-2 (hepatic stellate cells), and 3T3-L1 (adipocytes), and were not toxic to isolated human PBMCs and RBCs. Genotoxicity assessments found that SeNPs and ABE-SeNPs did not induce mutations in Salmonella typhimurium TA98 and TA100 (Ames test) as well as in Drosophila melanogaster (somatic mutation and recombination test). Noticeably, ABE-SeNPs inhibited mutation in TA98 and TA100 induced by AF-2, and in Drosophila induced by urethane, ethyl methanesulfonate, and mitomycin c, suggesting their anti-mutagenicity ability. This study provides data that support the safety and anti-genotoxicity properties of ABE-SeNPs for the further development of SeNPs-based food supplements.

Flavonoid profile of Anoectochilus roxburghii (Wall.) Lindl. Under short-term heat stress revealed by integrated metabolome, transcriptome, and biochemical analyses.

Global warming severely threatens plant growth, and could lead to yield reduction. Although findings suggest that flavonoids play important roles in biological process in plants, their response to heat stress in Anoectochilus roxburghii (Wall.) Lindl. remains unclear. Here, we aimed to examine the flavonoid profile of A. roxburghii under heat stress and assess the effect of exogenous application of quercetin on heat stress tolerance. Metabolome analysis showed that quercetin, tricetin, isorhamnetin, scutellarein, and 4',7-Isoflavandiol were the main upregulated flavonoids in A. roxburghii, based on variable importance in the projection >1 and with fold change >2. Determination of the concentrations of the flavonoids using a standard curve revealed that quercetin, kaempferol, and isorhamnetin contents increased by 8.24-, 7.55-, and 5.01-fold, respectively, during heat stress, whereas rutin concentration decreased from 83.04 to 80.89 mg/kg (dry weight). Additionally, transcriptome analysis indicated increased expression of several genes in flavonoid biosynthesis pathways, including phenylalanine ammonia-lyase and chalcone synthase. Moreover, exogenous application of quercetin improved the antioxidant capacity and physiological parameters, including photosynthetic rate and chlorophyll content, of A. roxburghii under heat stress. Overall, the flavonoid profile of A. roxburghii under short-term heat stress was characterized based on integrated metabolomic, transcriptomic, and biochemical analyses, providing new insights for improving the biological value of A. roxburghii.

Serum metabolome profiling, network pharmacology analysis, and experimental validation of Anoectochilus roxburghii in the treatment of carbon tetrachloride-induced liver injury.

Anoectochilus roxburghii (Wall.) Lindl. (AR) has been traditionally used to treat inflammatory diseases, but the specific mechanism underlying its hepatoprotective effect remains unclear. Here, serum metabolomics and network pharmacology were employed to investigate the hepatoprotective mechanism of AR. Thirty male Sprague-Dawley rats were divided into six groups: normal, model, positive, high-dose AR, middle-dose AR, and low-dose AR. The positive group received therapeutic doses of silibinin, whereas the AR-treated groups received different doses of AR extract once daily. After 10 days of intragastric administration, the rats were intraperitoneally injected with a 50% CCl4 olive oil solution (2 mL/kg) to induce liver injury. Serum and liver samples were obtained, and GC-MS was utilized to monitor changes in serum metabolome. The levels of alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, and hydrooxproline in serum significantly increased in the model group. On the contrary, AR-treated group showed a significant decrease in the levels of alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, and hydrooxproline. Histopathological observation also revealed that the extent of liver injury was alleviated in the AR-treated group. Fifty differential metabolites were identified, suggesting that AR may prevent liver damage by modulating carbohydrate and amino acid metabolism.

Effects of dietary supplementation of Anoectochilus roxburghii extract (ARE) on growth performance, abdominal fat deposition, meat quality, and gut microbiota in broilers.

The broiler industry frequently encounters 2 common problems: excessive deposition of abdominal fat and poor quality of meat. However, there are limited nutritional manipulation strategies to address these issues. While Anoectochilus roxburghii (Wall.) Lindl., a traditional Chinese herb, has been shown to have multiple beneficial effects in humans, its potential roles in broiler chickens remain unexplored. In this study, the effects of dietary supplementation with Anoectochilus roxburghii extract (ARE) on growth performance, abdominal fat deposition, meat quality, blood indices, and gut microbiota were investigated in yellow-feather broiler chickens. A total of 90 twenty-one-day-old yellow-feather broilers were randomly divided into 3 treatments, and each treatment included 5 replicates with 6 birds per replicate. Birds were fed a basal diet supplemented with 0, 0.15, or 0.30% ARE for 6 wk. The results showed that the inclusion of ARE in the diet did not have any significant effect on meat yield (P > 0.05). However, it did lead to a reduction in abdominal fat deposition and an improvement in meat quality (P < 0.05). Mechanistically, the addition of ARE inhibited lipid biosynthesis and enhanced lipid breakdown in both the liver and adipose tissue of the broilers. Furthermore, ARE supplementation increased the antioxidase activities in the muscle and serum of the broilers (P < 0.05). In addition, the supplementation of ARE optimized the diversity and composition of the cecal microbiota, particularly by lowering the ratio of Firmicutes to Bacteroidetes (P < 0.05). Moreover, the abundance of some bacteria that were positively correlated with abdominal fat deposition was reduced by ARE, and vice versa (P < 0.05). Collectively, the results suggest that ARE is a promising candidate as a feed additive for reducing abdominal fat deposition and improving meat quality in the broiler industry.

Novel insights into orchid mycorrhiza functioning from stable isotope signatures of fungal pelotons.

Stable isotope signatures of fungal sporocarps have been instrumental in identifying carbon gains of chlorophyllous orchids from a fungal source. Yet, not all mycorrhizal fungi produce macroscopic sporocarps and frequently fungi of different taxa occur in parallel in orchid roots. To overcome this obstacle, we investigated stable isotope signatures of fungal pelotons extracted from orchid roots and compared these data to the respective orchid and reference plant tissues. Anoectochilus sandvicensis and Epipactis palustris represented specialized or unspecialized rhizoctonia-associated orchids. Epipactis atrorubens and Epipactis leptochila are orchids considered ectomycorrhiza-associated with different preferences for Basidio- and Ascomycota. 13 C enrichment of rhizoctonia pelotons was minor compared with plant tissues and significantly lower than enrichments of pelotons from ectomycorrhizal Epipactis species. 15 N values of pelotons from E. leptochila and E. atrorubens showed similar patterns as known for respective sporocarps of ectomycorrhizal Ascomycota and Basidiomycota, however, with an offset towards lower 15 N enrichments and nitrogen concentrations. Our results suggest an explicit fungal nutrition source of orchids associated with ectomycorrhizal fungi, whereas the low 13 C enrichment in rhizoctonia-associated orchids and fungal pelotons hamper the detection of carbon gains from fungal partners. 15 N isotopic pattern of orchids further suggests a selective transfer of 15 N-enriched protein-nitrogen into orchids.

Combined analysis of transcriptomics and metabolomics revealed complex metabolic genes for diterpenoids biosynthesis in different organs of Anoectochilus roxburghii.

Objective: Diterpenoids with a wide variety of biological activities from Anoectochilus roxburghii, a precious medicinal plant, are important active components. However, due to the lack of genetic information on the metabolic process of diterpenoids in A. roxburghii, the genes involved in the molecular regulation mechanism of diterpenoid metabolism are still unclear. This study revealed the complex metabolic genes for diterpenoids biosynthesis in different organs of A. roxburghii by combining analysis of transcriptomics and metabolomics. Methods: The differences in diterpenoid accumulation in roots, stems and leaves of A. roxburghii were analyzed by metabonomic analysis, and its metabolic gene information was obtained by transcriptome sequencing. Then, the molecular mechanism of differential diterpenoid accumulation in different organs of A. roxburghii was analyzed from the perspective of gene expression patterns. Results: A total of 296 terpenoid metabolites were identified in the five terpenoid metabolic pathways in A. roxburghii. There were 38, 34, and 18 diterpenoids with different contents between roots and leaves, between leaves and stems, and between roots and stems, respectively. Twenty-nine metabolic enzyme genes with 883 unigenes in the diterpenoid synthesis process were identified, and the DXS and FDPS in the terpenoid backbone biosynthesis stage and CPA, GA20ox, GA3ox, GA2ox, and MAS in the diterpenoid biosynthesis stage were predicted to be the key metabolic enzymes for the accumulation of diterpenoids. In addition, 14 key transcription factor coding genes were predicted to be involved in the regulation of the diterpenoid biosynthesis. The expression of genes such as GA2ox, MAS, CPA, GA20ox and GA3ox might be activated by some of the 14 transcription factors. The transcription factor NTF-Y and PRE6 were predicted to be the most important transcription factors. Conclusion: This study determined 29 metabolic enzyme genes and predicted 14 transcription factors involved in the molecular regulation mechanism of diterpenoid metabolism in A. roxburghii, which provided a reference for the further study of the molecular regulation mechanism of the accumulation of diterpenoids in different organs of A. roxburghii.

Protective Effect of Anoectochilus formosanus Polysaccharide against Cyclophosphamide-Induced Immunosuppression in BALB/c Mice.

In this study, Anoectochilus formosanus polysaccharide (AFP) was acquired a via water extraction and alcohol precipitation method. The immunoregulatory activity of AFP was first evaluated on cyclophosphamide (Cy)-treated mice. Galacturonic acid, glucose and galactose were confirmed to be the main components of AFP. AFP demonstrated the ability to stimulate the production of TNF-α and IL-6 in RAW 264.7 macrophages. Not surprisingly, the activation of the NF-κB signaling pathway by AFP was validated via Western blot analysis. Furthermore, AFP could alleviate Cy-induced immunosuppression, and significantly enhance the immunity of mice via increasing the thymus index and body weight, stimulating the production of cytokines (IgA, IgG, SIgA, IL-2, IL-6 and IFN-γ). The improvement in the intestinal morphology of immunosuppressed mice showed that AFP could alleviate Cy-induced immune toxicity. These results have raised the possibility that AFP may act as a natural immunomodulator. Overall, the study of AFP was innovative and of great significance for AFP's further application and utilization.

Polysaccharide and ethanol extracts of Anoectochilus formosanus Hayata: Antioxidant, wound-healing, antibacterial, and cytotoxic activities.

Introduction: Polysaccharide and alcohol extracts of Anoectochilus formosanus Hayata have attracted great attention as they exhibit noteworthy properties such as prebiotic and anti-hyperglycemic effects. However, the antioxidant and wound-healing activities of the polysaccharide extract as well as the antibacterial and cytotoxic effects of the ethanol extracts have not been thoroughly uncovered. Therefore, our study investigated these bioactivities of the two extracts prepared from Anoectochilus formosanus to broaden understandings of medical benefits of the plant. Methods: The monosaccharide composition was analyzed by HPAEC-PAD. The antioxidant and wound-healing activities of the polysaccharide extract were evaluated by ABTS and scratch assays, respectively. The broth dilution method was used to determine the antibacterial ability of the ethanol extract. Additionally, the cytotoxic and mechanistic effects of this extract against hepatocellular carcinoma HUH-7 cells was assessed by MTT assay, qRT-PCR and Western blotting methods. Results: The polysaccharide extract possessed an effective free radical scavenging ability in an ABTS assay (IC50 = 44.92 µg/ml). The extract also ameliorated wound recovery in a fibroblast scratch assay. Meanwhile, the ethanol extract was able to inhibit the growth of Staphylococcus aureus (MIC = 2500 µg/ml), Bacillus cereus (MIC = 2500 µg/ml), Escherichia coli (MIC = 2500 µg/ml), and Pseudomonas aeruginosa (MIC = 1250 µg/ml). Additionally, it repressed the viability of HUH-7 cells (IC50 = 53.44 µg/ml), possibly through upregulating the expression of caspase 3 (CASP3), CASP8, and CASP9 at both mRNA and protein levels. Conclusion: The polysaccharide extract of A. formosanus exhibited the antioxidant and wound-healing properties whereas the ethanol extract showed the antibacterial activity and cytotoxicity against HUH-7 cells. These findings specify notable biological effects of the two extracts which could be of potential use in human healthcare.

Isolation, structural and bioactivities of polysaccharides from Anoectochilus roxburghii (Wall.) Lindl.: A review.

Anoectochilus roxburghii (Wall.) Lindl. (A. roxburghii), a valuable herbal medicine in China, has great medicinal and edible value. Polysaccharides, as one of the main active components of A. roxburghii, comprise glucose, arabinose, xylose, galactose, rhamnose, and mannose in different molar ratios and glycosidic bond types. By varying the sources and extraction methods of A. roxburghii polysaccharides (ARPS), different structural characteristics and pharmacological activities can be elucidated. ARPS has been reported to exhibit antidiabetic, hepatoprotective, anti-inflammatory, antioxidant, antitumor, and immune regulation activities. This review summarizes the available literature on the extraction and purification methods, structural features, biological activities, and applications of ARPS. The shortcomings of the current research and potential focus in future studies are also highlighted. This review provides systematic and current information on ARPS to promote their further exploitation and application.

Structural characterization and antinociceptive activity of polysaccharides from Anoectochilus elatus.

Anoectochilus elatus is a new record species from Yunnan province in China discovered by our group in 2018, used in folk as the most popular Anoectochilus species A. roxburghii for medicinal and culinary purposes. The crude polysaccharide of Anoectochilus elatus (AEP) exhibited significant antinociceptive effects against both chemical and thermal nociception in vivo. Bio-guided isolation identified GJXL-1 as the leading analgesic polysaccharide in AEP. Detailed structural analyses rationalized GJXL-1 (molecular weight: 10.3 kDa) as an α-D-1,4-linked glucan unexpectedly branched at O-3, and O-6 position. GJXL-1 dose-dependently suppressed acetic acid-induced writhing in mice and decreased the serum levels of NO, IL-6 and TNF-α, which also repressed the licking times in both the first and second phases in formalin test. Furthermore, only L-nitroarginine partly reversed the analgesic activity of GJXL-1, indicating that GJXL-1's efficacy was partially mediated by NO regulation, possibly through inhibiting IRAK4/TAK1/NF-κB signaling pathway, and modulating gut microbiota and short-chain fatty acids production. In addition, the motor impairment and hypnotic effects of GJXL-1 were excluded. Our study suggests that GJXL-1 can be regarded as a promising and safe drug candidate for diverse pain disorders, and also a promising prebiotic candidate to maintain intestinal homeostasis and promote human gut health.