Isoorientin Inhibits Amyloid β25–35-Induced Neuronal Inflammation in BV2 Cells by Blocking the NF-κB Signaling Pathway

Alzheimer’s disease (AD) is a severe neurodegenerative disorder. AD is pathologically characterized by the formation of intracellular neurofibrillary tangles, and extracellular amyloid plaques which were comprised of amyloid-beta (Aβ) peptides. Aβ induces neurodegeneration by activating microglia, which triggers neurotoxicity by releasing various inflammatory mediators and reactive oxygen species (ROS). Nuclear factor-kappa B (NF-κB) is expressed in human tissues including the brain and plays an important role in Aβ-mediated neuronal inflammation. Thus, the identification of molecules that inhibit the NF-κB pathway is considered an attractive strategy for the treatment and prevention of AD. Isoorientin (3′,4′,5,7-Tetrahydroxy-6-C-glucopyranosyl flavone; ISO), which can be extracted from several plant species, such as Philostachys and Patrinia is known to have various pharmacological activities such as anticancer, antioxidant, and antibacterial activity. However, the effect of ISO on Aβ-mediated inflammation and apoptosis in the brain has yet to be elucidated. In the present study, we investigated whether ISO regulated Aβ-induced neuroinflammation in microglial cells and further explored the underlying mechanisms. Our results showed that ISO inhibited the expression of iNOS and COX-2 induced by Aβ_25–35. And, it inhibited the secretion of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). In addition, ISO reduced the ROS production in Aβ_25–35-induced BV2 cells and inhibited NF-κB activation. Furthermore, ISO blocked Aβ_25–35-induced apoptosis of BV2 cells. Based on these findings, we suggest that ISO represents a promising therapeutic drug candidate for the treatment and prevention of AD.     

Honokiol ameliorates endothelial dysfunction through suppression of PTX3 expression,a key mediator of IKK/IκB/NF-κB,in atherosclerotic cell model

Pentraxin 3 (PTX3) was identified as a marker of the inflammatory response and overexpressed in various tissues and cells related to cardiovascular disease. Honokiol, an active component isolated from the Chinese medicinal herb Magnolia officinalis, was shown to have a variety of pharmacological activities. In the present study, we aimed to investigate the effects of honokiol on palmitic acid (PA)-induced dysfunction of human umbilical vein endothelial cells (HUVECs) and to elucidate potential regulatory mechanisms in this atherosclerotic cell model. Our results showed that PA significantly accelerated the expression of PTX3 in HUVECs through the IκB kinase (IKK)/IκB/nuclear factor-κB (NF-κB) pathway, reduced cell viability, induced cell apoptosis and triggered the inflammatory response. Knockdown of PTX3 supported cell growth and prevented apoptosis by blocking PA-inducted nitric oxide (NO) overproduction. Honokiol significantly suppressed the overexpression of PTX3 in PA-inducted HUVECs by inhibiting IκB phosphorylation and the expression of two NF-κB subunits (p50 and p65) in the IKK/IκB/NF-κB signaling pathway. Furthermore, honokiol reduced endothelial cell injury and apoptosis by regulating the expression of inducible NO synthase and endothelial NO synthase, as well as the generation of NO. Honokiol showed an anti-inflammatory effect in PA-inducted HUVECs by significantly inhibiting the generation of interleukin-6 (IL-6), IL-8 and monocyte chemoattractant protein-1. In summary, honokiol repaired endothelial dysfunction by suppressing PTX3 overexpression in an atherosclerotic cell model. PTX3 may be a potential therapeutic target for atherosclerosis.     

Decreased Human Platelet Activation and Mouse Pulmonary Thrombosis by Rutaecarpine and Comparison of the Relative Effectiveness with BAY11-7082: Crucial Signals of p38-NF-κB

Platelets play a critical role in arterial thrombosis. Rutaecarpine (RUT) was purified from Tetradium ruticarpum, a well-known Chinese medicine. This study examined the relative activity of RUT with NF-κB inhibitors in human platelets. BAY11-7082 (an inhibitor of IκB kinase [IKK]), Ro106-9920 (an inhibitor of proteasomes), and RUT concentration-dependently (1–6 μM) inhibited platelet aggregation and P-selectin expression. RUT was found to have a similar effect to that of BAY11-7082; however, it exhibits more effectiveness than Ro106-9920. RUT suppresses the NF-κB pathway as it inhibits IKK, IκBα, and p65 phosphorylation and reverses IκBα degradation in activated platelets. This study also investigated the role of p38 and NF-κB in cell signaling events and found that SB203580 (an inhibitor of p38) markedly reduced p38, IKK, and p65 phosphorylation and reversed IκBα degradation as well as p65 activation in a confocal microscope, whereas BAY11-7082 had no effects in p38 phosphorylation. The 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay shows that RUT and BAY11-7082 did not exhibit free radical scavenging activity. In the in vivo study, compared with BAY11-7082, RUT more effectively reduced mortality in adenosine diphosphate (ADP)-induced acute pulmonary thromboembolism without affecting the bleeding time. In conclusion, a distinctive pathway of p38-mediated NF-κB activation may involve RUT-mediated antiplatelet activation, and RUT could act as a strong prophylactic or therapeutic drug for cardiovascular diseases.     

Effect of Korean Red Ginseng and Rg3 on Asian Sand Dust-Induced MUC5AC,MUC5B,and MUC8 Expression in Bronchial Epithelial Cells

Korean Red ginseng (KRG), commonly used in traditional medicine, has anti-inflammatory, anti- oxidative, and anti-tumorigenic properties. Asian sand dust (ASD) is known to aggravate upper and lower airway inflammatory responses. BEAS-2B cells were exposed to ASD with or without KRG or ginsenoside Rg3. Mucin 5AC (MUC5AC), MUC5B, and MUC8 mRNA and protein expression levels were determined using quantitative RT-PCR and enzyme-linked immunosorbent assay. Nuclear factor kappa B (NF-κB), activator protein 1, and mitogen-activated protein kinase expression and activity were determined using western blot analysis. ASD induced MUC5AC, MUC5B, and MUC8 mRNA and protein expression in BEAS-2B cells, which was significantly inhibited by KRG and Rg3. Although ASD-induced mucin expression was associated with NF-κB and p38 mitogen-activated protein kinase (MAPK) activity, KRG and Rg3 significantly suppressed only ASD-induced NF-κB expression and activity. KRG and Rg3 inhibited ASD-induced mucin gene expression and protein production from bronchial epithelial cells. These results suggest that KRG and Rg3 have potential for treating mucus-producing airway inflammatory diseases.     

Rosmanol and Carnosol Synergistically Alleviate Rheumatoid Arthritis through Inhibiting TLR4/NF-κB/MAPK Pathway

Callicarpalongissima has been used as a Yao folk medicine to treat arthritis for years in China, although its active anti-arthritic moieties have not been clarified so far. In this study, two natural phenolic diterpenoids with anti-rheumatoid arthritis (RA) effects, rosmanol and carnosol, isolated from the medicinal plant were reported on for the first time. In type II collagen-induced arthritis DBA/1 mice, both rosmanol (40 mg/kg/d) and carnosol (40 mg/kg/d) alone alleviated the RA symptoms, such as swelling, redness, and synovitis; decreased the arthritis index score; and downregulated the serum pro-inflammatory cytokine levels of interleukin 6 (IL-6), monocyte chemotactic protein 1 (MCP-1), and tumor necrosis factor α (TNF-α). Additionally, they blocked the activation of the Toll-like receptor 4 (TLR4)/nuclear factor κB (NF-κB)/c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) pathways. Of particular interest was that when they were used in combination (20 mg/kg/d each), the anti-RA effect and inhibitory activity on the TLR4/NF-κB/MAPK pathway were significantly enhanced. The results demonstrated that rosmanol and carnosol synergistically alleviated RA by inhibiting inflammation through regulating the TLR4/NF-κB/MAPK pathway, meaning they have the potential to be developed into novel, safe natural combinations for the treatment of RA.     

Polyphenols isolated from Broussonetia kazinoki prevent cytokine-induced β-cell damage and the development of type 1 diabetes

The axis of nuclear factor κB (NF-κB)-inducible NO synthase (iNOS)-nitric oxide plays a key role in cytokine- and streptozotocin-mediated pancreatic β-cell damage. In this study, we investigated the effects of kazinol C and isokazinol D isolated from Broussonetia kazinoki on the β-cell viability and function. RINm5F cells and primary islets were used for in vitro and ex vivo cytokine toxicity experiments, respectively. For type 1 diabetes induction, mice were injected with multiple low-dose streptozotocin (MLDS). Cytokine-induced toxicity was completely abolished in both RINm5F cells and islets that were pretreated with either kazinol C or isokazinol D. Both kazinols inhibited the NF-κB signaling pathway, thereby inhibiting cytokine-mediated iNOS induction, nitric oxide production, apoptotic cell death and defects in insulin secretion. Moreover, the occurrence of diabetes in MLDS-treated mice was efficiently attenuated in kazinol-pretreated mice. Immunohistochemical analysis revealed that the numbers of terminal deoxynucleotidyl transferase dUTP nick end labeling-positive apoptotic cells and nuclear p65-positive cells were significantly decreased in kazinol-pretreated mice. Our results suggest that kazinol C and isokazinol D block the NF-κB pathway, thus reducing the extent of β-cell damage. Therefore, kazinol C and isokazinol D may have therapeutic value in delaying pancreatic β-cell damage in type 1 diabetes.     

Sensitization to Drug Treatment in Precursor B-Cell Acute Lymphoblastic Leukemia Is Not Achieved by Stromal NF-κB Inhibition of Cell Adhesion but by Stromal PKC-Dependent Inhibition of ABC Transporters Activity

Cell adhesion to stromal support and the associated intracellular signaling are central to drug resistance, therefore blocking both has been effective in increasing drug sensitization in leukemia. The stromal Ser/Thr protein kinase C (PKC) has been found to be important for conferring protection to leukemic cells. We aimed at elucidating the intracellular signals connected to cell adhesion and to stromal PKC. We found that NF-κB and Akt were up-regulated in mesenchymal stem cells (MSC) after binding of B-cell acute lymphoblastic leukemia (B-ALL) cells. Nevertheless, Akt inhibition did not induce B-ALL cell detachment. In spite of a clear activation of the NF-κB signaling pathway after B-ALL cell binding (up-regulation NF-κB1/2, and down-regulation of the IKBε and IKBα inhibitors) and an important reduction in cell adhesion after NF-κB inhibition, sensitization to the drug treatment was not observed. This was opposite to the PKC inhibitors Enzastaurin and HKPS, a novel chimeric peptide inhibitor, that were able to increase sensitization to dexamethasone, methotrexate, and vincristine. PLCγ1, Erk1/2, and CREB appear to be related to PKC signaling and PKC effect on drug sensitization since they were contra-regulated by HKPS when compared to dexamethasone-treated cells. Additionally, PKC inhibition by HKPS, but not by Enzastaurin, in MSC reduced the activity of three ABC transporters in leukemic cells treated with dexamethasone, a new indirect mechanism to increase sensitization to drug treatment in B-ALL cells. Our results show the validity of targeting the functional characteristic acquired and modulated during cell-to-cell interactions occurring in the leukemic niche.     

Diterpenoid Compounds Isolated from Chloranthus oldhamii Solms Exert Anti-Inflammatory Effects by Inhibiting the IKK/NF-κB Pathway

Chloranthus oldhamii Solms (CO) is a folk medicine for treating infection and arthritis pain but its pharmacological activity and bioactive compounds remain mostly uncharacterized. In this study, the anti-inflammatory compounds of C. oldhamii were identified using an LPS-stimulated, NF-κB-responsive RAW 264.7 macrophage reporter line. Three diterpenoid compounds, 3α-hydroxy-ent-abieta-8,11,13-triene (CO-9), 3α, 7β-dihydroxy-ent-abieta-8,11,13-triene (CO-10), and decandrin B (CO-15) were found to inhibit NF-κB activity at nontoxic concentrations. Moreover, CO-9 and CO-10 suppressed the expression of IL-6 and TNF-α in LPS-stimulated RAW 264.7 cells. The inhibitory effect of CO-9 on TNF-α and IL-6 expression was further demonstrated using LPS-treated bone marrow-derived macrophages. Furthermore, CO-9, CO-10, and CO-15 suppressed LPS-triggered COX-2 expression and downstream PGE2 production in RAW 264.7 cells. CO-9 and CO-10 also reduced LPS-triggered iNOS expression and nitrogen oxide production in RAW 264.7 cells. The anti-inflammatory mechanism of the most effective compound, CO-9, was further investigated. CO-9 attenuated LPS-induced NF-κB activation by reducing the phosphorylation of IKKα/β (Ser176/180), IκBα (Ser32), and p65 (Ser534). Conversely, CO-9 did not affect the LPS-induced activation of MAPK signaling pathways. In summary, this study revealed new anti-inflammatory diterpenoid compounds from C. oldhamii and demonstrated that the IKK-mediated NK-κB pathway is the major target of these compounds.     

The In Vitro Anti-Inflammatory Activities of Galangin and Quercetin towards the LPS-Injured Rat Intestinal Epithelial (IEC-6) Cells as Affected by Heat Treatment

Flavonols possess several beneficial bioactivities in vitro and in vivo. In this study, two flavonols galangin and quercetin with or without heat treatment (100 °C for 15–30 min) were assessed for their anti-inflammatory activities in lipopolysaccharide (LPS)-stimulated rat intestinal epithelial (IEC-6) cells and whether the heat treatment caused activity changes. The flavonol dosages of 2.5–20 μmol/L had no cytotoxicity on the cells but could enhance cell viability (especially using 5 μmol/L flavonol dosage). The flavonols could decrease the production of prostaglandin E₂ and three pro-inflammatory cytokines interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-α, and simultaneously promote the production of two anti-inflammatory cytokines IL-10 and transforming growth factor-β. The Western-blot results verified that the flavonols could suppress the LPS-induced expression of TLR4 and phosphorylated IκBα and p65, while the molecular docking results also illustrated that the flavonols could bind with TLR4 and NF-κB to yield energy decreases of −(21.9–28.6) kJ/mol. Furthermore, an inhibitor BAY 11-7082 blocked the NF-κB signaling pathway by inhibiting the expression of phosphorylated IκBα/p65 and thus mediated the production of IL-6/IL-10 as the flavonols did, which confirmed the assessed anti-inflammatory effect of the flavonols. Consistently, galangin had higher anti-inflammatory activity than quercetin, while the heated flavonols (especially those with longer heat time) were less active than the unheated counterparts to exert these target anti-inflammatory effects. It is highlighted that the flavonols could antagonize the LPS-caused IEC-6 cells inflammation via suppressing TLR4/NF-κB activation, but heat treatment of the flavonols led to reduced anti-inflammatory efficacy.     

Blockade of NF-κB Translocation and of RANKL/RANK Interaction Decreases the Frequency of Th2 and Th17 Cells Capable of IL-4 and IL-17 Production,Respectively, in a Mouse Model of Allergic Asthma

The main purpose of this study was to investigate whether the blockade of the interaction between the receptor activator of nuclear factor-κB (NF-ĸB) ligand (RANKL) and its receptor RANK as well as the blockade of NF-κB inhibitor kinase (IKK) and of NF-κB translocation have the potential to suppress the pathogenesis of allergic asthma by inhibition and/or enhancement of the production by CD4⁺ and CD8⁺ T cells of important cytokines promoting (i.e., IL-4 and IL-17) and/or inhibiting (i.e., IL-10 and TGF-β), respectively, the development of allergic asthma. Studies using ovalbumin(OVA)-immunized mice have demonstrated that all the tested therapeutic strategies prevented the OVA-induced increase in the absolute number of IL-4- and IL-17-producing CD4⁺ T cells (i.e., Th2 and Th17 cells, respectively) indirectly, i.e., through the inhibition of the clonal expansion of these cells in the mediastinal lymph nodes. Additionally, the blockade of NF-κB translocation and RANKL/RANK interaction, but not IKK, prevented the OVA-induced increase in the percentage of IL-4-, IL-10- and IL-17-producing CD4⁺ T cells. These latter results strongly suggest that both therapeutic strategies can directly decrease IL-4 and IL-17 production by Th2 and Th17 cells, respectively. This action may constitute an important mechanism underlying the anti-asthmatic effect induced by the blockade of NF-κB translocation and of RANKL/RANK interaction. Thus, in this context, both these therapeutic strategies seem to have an advantage over the blockade of IKK. None of the tested therapeutic strategies increased both the absolute number and frequency of IL-10- and TGF-β-producing Treg cells, and hence they lacked the potential to inhibit the development of the disease via this mechanism.     

Anthocyanins Derived from Vitis coignetiae Pulliat Contributes Anti-Cancer Effects by Suppressing NF-κB Pathways in Hep3B Human Hepatocellular Carcinoma Cells and In Vivo

We previously demonstrated that anthocyanins from the fruits of Vitis coignetiae Pulliat (AIMs) induced the apoptosis of hepatocellular carcinoma cells. However, many researchers argued that the concentrations of AIMs were too high for in vivo experiments. Therefore, we performed in vitro at lower concentrations and in vivo experiments for the anti-cancer effects of AIMs. AIMs inhibited the cell proliferation of Hep3B cells in a dose-dependent manner with a maximum concentration of 100 µg/mL. AIMs also inhibited the invasion and migration at 100 µg/mL concentration with or without the presence of TNF-α. To establish the relevance between the in vitro and in vivo results, we validated their effects in a Xenograft model of Hep3B human hepatocellular carcinoma cells. In the in vivo test, AIMs inhibited the tumorigenicity of Hep3B cells in the xenograft mouse model without showing any clinical signs of toxicity or any changes in the body weight of mice. AIMs inhibited the activation NF-κB and suppressed the NF-κB-regulated proteins, intra-tumoral microvessel density (IMVD) and the Ki67 activity of Hep3B xenograft tumors in athymic nude mice. In conclusion, this study indicates that AIMs have anti-cancer effects (inhibition of proliferation, invasion, and angiogenesis) on human hepatocellular carcinoma xenograft through the inhibition of NF-κB and its target protein.     

Caragana rosea Turcz Methanol Extract Inhibits Lipopolysaccharide-Induced Inflammatory Responses by Suppressing the TLR4/NF-κB/IRF3 Signaling Pathways

Caragana rosea Turcz, which belongs to the Leguminosae family, is a small shrub found in Northern and Eastern China that is known to possess anti-inflammatory properties and is used to treat fever, asthma, and cough. However, the underlying molecular mechanisms of its anti-inflammatory effects are unknown. Therefore, we used lipopolysaccharide (LPS) in RAW264.7 macrophages to investigate the molecular mechanisms that underlie the anti-inflammatory activities of a methanol extract of Caragana rosea (Cr-ME). We showed that Cr-ME reduced the production of nitric oxide (NO) and mRNA levels of iNOS, TNF-α, and IL-6 in a concentration-dependent manner. We also found that Cr-ME blocked MyD88- and TBK1-induced NF-κB and IRF3 promoter activity, suggesting that it affects multiple targets. Moreover, Cr-ME reduced the phosphorylation levels of IκBα, IKKα/β and IRF3 in a time-dependent manner and regulated the upstream NF-κB proteins Syk and Src, and the IRF3 protein TBK1. Upon overexpression of Src and TBK1, Cr-ME stimulation attenuated the phosphorylation of the NF-κB subunits p50 and p65 and IRF3 signaling. Together, our results suggest that the anti-inflammatory activity of Cr-ME occurs by inhibiting the NF-κB and IRF3 signaling pathways.     

Mitigating Effect of Lindera obtusiloba Blume Extract on Neuroinflammation in Microglial Cells and Scopolamine-Induced Amnesia in Mice

Lindera obtusiloba Blume (family, Lauraceae), native to Northeast Asia, has been used traditionally in the treatment of trauma and neuralgia. In this study, we investigated the neuroinflammatory effect of methanol extract of L. obtusiloba stem (LOS-ME) in a scopolamine-induced amnesia model and lipopolysaccharide (LPS)-stimulated BV2 microglia cells. LOS-ME downregulated the expression of inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2, inflammatory cytokines, and inhibited the phosphorylation of nuclear factor kappa-B (NF-ĸB) and extracellular signal-regulated kinase (ERK) in LPS-stimulated BV2 cells. Male C57/BL6 mice were orally administered 20 and 200 mg/kg of LOS-ME for one week, and 2 mg/kg of scopolamine was administered intraperitoneally on the 8th day. In vivo behavioral experiments (Y-maze and Morris water maze test) confirmed that LOS-ME alleviated cognitive impairments induced by scopolamine and the amount of iNOS expression decreased in the hippocampus of the mouse brain. Microglial hyper-activation was also reduced by LOS-ME pretreatment. These findings suggest that LOS-ME might have potential in the treatment for cognitive improvement by regulating neuroinflammation.     

Novel c-Jun N-Terminal Kinase (JNK) Inhibitors with an 11H-Indeno[1,2-b]quinoxalin-11-one Scaffold

c-Jun N-terminal kinase (JNK) plays a central role in stress signaling pathways implicated in important pathological processes, including rheumatoid arthritis and ischemia-reperfusion injury. Therefore, inhibition of JNK is of interest for molecular targeted therapy to treat various diseases. We synthesized 13 derivatives of our reported JNK inhibitor 11H-indeno[1,2-b]quinoxalin-11-one oxime and evaluated their binding to the three JNK isoforms and their biological effects. Eight compounds exhibited submicromolar binding affinity for at least one JNK isoform. Most of these compounds also inhibited lipopolysaccharide (LPS)-induced nuclear factor-κB/activating protein 1 (NF-κB/AP-1) activation and interleukin-6 (IL-6) production in human monocytic THP1-Blue cells and human MonoMac-6 cells, respectively. Selected compounds (4f and 4m) also inhibited LPS-induced c-Jun phosphorylation in MonoMac-6 cells, directly confirming JNK inhibition. We conclude that indenoquinoxaline-based oximes can serve as specific small-molecule modulators for mechanistic studies of JNKs, as well as potential leads for the development of anti-inflammatory drugs.     

Ganoderma species discrimination by dual-mode chromatographic fingerprinting: A study on stationary phase effects in hydrophilic interaction chromatography and reduction of sample misclassification rate by additional use of reversed-phase chromatography

Acetonitrile–water extracts of several Ganoderma species – a mushroom being used in Traditional Chinese Medicine – were analysed by liquid chromatography–UV detection in hydrophilic interaction chromatography (HILIC) and reversed-phase (RP) elution modes. A set of six polar stationary phases was used for HILIC runs. These columns had remarkably different separation properties under binary gradient conditions as evinced by hierarchical cluster analysis on retention patterns of seven test compounds. Complementary measurements of RP chromatograms were carried out on a C₁₈ packing. Injection precision (n = 5) and intra-day precision (n = 5) were each <2.0% RSD (HILIC) and <0.7% RSD (RP) for relative retention times of main characteristic peaks of a sample extract while for relative peak areas RSD values were max. 6.8%. Repetitive analysis (n = 7) of a processed sample stored in the autosampler tray for 48 h was used to confirm within-sequence sample stability. Eleven Ganoderma lucidum samples served as training set for the construction of column-specific simulated mean chromatograms. Validation with twelve samples comprising G. lucidum, Ganoderma sinense, Ganoderma atrum, and Ganoderma tsugae by correlation coefficient based similarity evaluation of peak patterns showed that a discrimination of G. lucidum from other Ganoderma species by means of chromatographic fingerprints is conceptually possible on all columns, except of a bare silica packing. The importance of the combined use of RP and HILIC fingerprints to improve the rate of correct sample classification was demonstrated by the fact that each one G. sinense specimen was wrongly assigned being G. lucidum by all HILIC fingerprints but not the RP fingerprint and vice versa. The present data revealed that (i) the analysis of complex biological materials by quasi orthogonal chromatographic modes such as HILIC and RP may deliver more discriminative information than single-mode approaches which strengthens the reliability of fingerprint-based sample classification and (ii) different retention and selectivity characteristics of polar bonded silica packings in the HILIC elution mode may only have a minor impact on chemometric sample discrimination capabilities in such kind of pattern-oriented metabolomics separation problems.     

Purification and Characterization of Antibacterial Activity against Phytopathogenic Bacteria in Culture Fluids from Ganoderma lucidum

Previous studies of Ganoderma lucidum have focused on its medicinal applications. Limited information is available about its antibacterial activity against plant pathogens. Thus, the goal of this study was to purify and characterize the antibacterial activity against plant pathogenic bacteria from culture fluids of G. lucidum. The nature of the bioactive components was determined using heat boiling, organic solvents, dialysis tubing, gel exclusion chromatography (GEC), proteinase sensitivity, HPLC, HPLC-APCI-MS, and GC-MS. The bioactive compounds were neither lipid, based on their solubility, nor proteic in nature, based on proteinase digestion and heat stability. The putative-bioactive polysaccharides have molecular weights that range from 3500 to 4500 Daltons as determined by dialysis tubing, GEC and APCI-MS analysis. The composition of the antibacterial compounds was determined by GC-MS. This is the first report of small polysaccharides produced by G. lucidum with activity against bacterial plant pathogens.     

Improved Method of Extraction and High-Performance Liquid Chromatographic Separation of Ganoderic Acids from Ganoderma Lucidum

A rapid and efficient method is described for the extraction of ganoderic acids from Ganodemia lucidum by means of a small Sep-Pak C₁₈ cartridge. The application of high-performance liquid chromatography to determine the chemical compositions of strains of Ganoderma lucidum is also reported.