Inotodiol, an antiasthmatic agent with efficacy and safety, preferentially impairs membrane-proximal signaling for mast cell activation.

While inhaled corticosteroids (ICSs) are the mainstay of asthma treatment, due to compliance, drug safety, and resistance issues, new medications to replace ICSs are in high demand. Inotodiol, a fungal triterpenoid, showed a unique immunosuppressive property with a preference for mast cells. It exerted a mast cell-stabilizing activity equally potent to dexamethasone in mouse anaphylaxis models when orally administered in a lipid-based formulation, upgrading bioavailability. However, it was four to over ten times less effective in suppressing other immune cell subsets, depending on the subsets, than dexamethasone showing invariably potent inhibition. Accordingly, inotodiol affected the membrane-proximal signaling for activating mast cell functions more profoundly than other subsets. Inotodiol also effectively prevented asthma exacerbation. Importantly, considering the no-observed-adverse-effect level of inotodiol was over 15 times higher than dexamethasone, its therapeutic index would be at least eight times better,implying that inotodiol is a viable option for replacing CSs in treating asthma.

A Novel Bioanalytical Method for Determination of Inotodiol Isolated from Inonotus Obliquus and Its Application to Pharmacokinetic Study.

In this study, we developed a bioanalytical method using liquid chromatography coupled to triple quadrupole tandem mass spectrometry (LC-MS/MS) to apply to a pharmacokinetic study of inotodiol, which is known for its anti-cancer activity. Plasma samples were prepared with alkaline hydrolysis, liquid-liquid extraction, and solid-phase extraction. Inotodiol was detected in positive mode with atmospheric pressure chemical ionization by multiple-reaction monitoring mode using LC-MS/MS. The developed method was validated with linearity, accuracy, and precision. Accuracy ranged from 97.8% to 111.9%, and the coefficient of variation for precision was 1.8% to 4.4%. The developed method was applied for pharmacokinetic study, and the mean pharmacokinetic parameters administration were calculated as follows: λz 0.016 min-1; T1/2 49.35 min; Cmax 2582 ng/mL; Cl 0.004 ng/min; AUC0-t 109,500 ng×min/mL; MRT0-t 32.30 min; Vd 0.281 mL after intravenous administration at dose of 2 mg/kg and λz 0.005 min-1; T1/2 138.6 min; Tmax 40 min; Cmax 49.56 ng/mL; AUC0-t 6176 ng×in/mL; MRT0-t 103.7 min after oral administration. The absolute oral bioavailability of inotodiol was 0.45%, similar to nonpolar phytosterols. Collectively, this is the first bioanalytical method and pharmacokinetic study for inotodiol.

An Ex vivo Mast Cell Degranulation Assay using Crude Peritoneal Exudate Cells and Natural Antigen Stimulation.

Mast cell stabilizers are an essential part of allergy medication. Passive systemic anaphylaxis (PSA) is an animal assay widely used for investigating the effect of a pharmacological agent of interest on mast cells in vivo. As the anaphylactic symptoms are primarily attributed to exocytosis of the granules from mast cells, it is conceived that the agent to cause amelioration of the symptoms has a mast cell stabilizing activity. Despite the fact, it is prudent to confirm the activity by directly demonstrating the decline in the functional activity of mast cells following its treatment. In vitro degranulation assays using an immortalized mast cell line or cultured primary mast cells are routinely employed to that end. The results from the in vitro and in vivo assays may not always be akin to each other; however, as treatment conditions (e.g., treatment dose, time, surrounding environments) for the in vitro assays are often distinct from those for the in vivo assay such as PSA. In pursuit of an in vitro (or ex vivo) assay to reflect more closely the effect of a pharmacological agent on mast cells in vivo, we devised the ex vivo mast cell degranulation assay in which crude peritoneal exudate cells (PECs) isolated from the mice, treated with the agent and administered anti-dinitrophenol (DNP) IgE, were incubated directly with DNP on a carrier protein. It turned out that the assay was not only useful in validating the mast cell stabilizing activity of a pharmacological agent indicated by the in vivo assay but also practical and highly reproducible.

Chemical constituents of Vietnamese mangrove Hibiscus tiliaceus with antioxidant and alpha-glucosidase inhibitory activity.

Using various chromatographic techniques, a total of 15 compounds, including one novel megastigmane named tiliaceic acid A (1) and 14 known compounds, were isolated from the traditional medicinal Vietnamese mangrove Hibiscus tiliaceus. Their structures were confirmed based on spectroscopic experiments including, UV, 1 D- and 2 D-NMR, HR-ESI-MS, and ECD analysis. The antioxidant and α-glucosidase inhibitory activities of the isolated compounds from H. tiliaceus were evaluated for the first time. Compound 2 showed strong α-glucosidase inhibitory activity with an IC50 of 77.78 ± 1.00 µM compared with the positive control acarbose at 105.71 ± 2.29 µM.

Enhancement of an In Vivo Anti-Inflammatory Activity of Oleanolic Acid through Glycosylation Occurring Naturally in Stauntonia hexaphylla.

Stauntonia hexaphylla (Lardizabalaceae) has been used as a traditional herbal medicine in Korea and China for its anti-inflammatory and analgesic properties. As part of a bioprospecting program aimed at the discovery of new bioactive compounds from Korean medicinal plants, a phytochemical study of S. hexaphylla leaves was carried out leading to isolation of two oleanane-type triterpene saponins, 3-O-[ß-d-glucopyranosyl (1→2)-α-l-arabinopyranosyl] oleanolic acid-28-O-[ß-d-glucopyranosyl (1→6)-ß-d-glucopyranosyl] ester (1) and 3-O-α-l-arabinopyranosyl oleanolic acid-28-O-[ß-d-glucopyranosyl (1→6)-ß-d-glucopyranosyl] ester (2). Their structures were established unambiguously by spectroscopic methods such as one- and two-dimensional nuclear magnetic resonance and infrared spectroscopies, high-resolution electrospray ionization mass spectrometry and chemical reactions. Their anti-inflammatory activities were examined for the first time with an animal model for the macrophage-mediated inflammatory response as well as a cell-based assay using an established macrophage cell line (RAW 264.7) in vitro. Together, it was concluded that the saponin constituents, when they were orally administered, exerted much more potent activities in vivo than their sapogenin core even though both the saponins and the sapogenin molecule inhibited the RAW 264.7 cell activation comparably well in vitro. These results imply that saponins from S. hexaphylla leaves have a definite advantage in the development of oral medications for the control of inflammatory responses.

Anti-allergic effect of inotodiol, a lanostane triterpenoid from Chaga mushroom, via selective inhibition of mast cell function.

Inotodiol is a lanostane triterpenoid found only in Chaga mushroom. In the previous study investigating anti-allergic effects of fractionated Chaga mushroom extracts, we have found evidence that purified inotodiol holds an activity to suppress the mast cell function in vivo. To address the therapeutic relevance of the finding, in this study, we investigated whether inotodiol could also alleviate allergy symptoms observed in a chicken ovalbumin (cOVA)-induced mouse model of food allergy. Like the crude 70% ethanol extract of Chaga mushroom (320 mg/kg), oral administration of inotodiol (20 mg/kg), regardless of whether that was for preventive or treatment purpose, resulted in a significant improvement in allergic symptoms and inflammatory lesions in the small intestine appearing after repeated oral challenge with cOVA. Despite the results that inotodiol (20 mg/kg) and the Chaga mushroom extract (320 mg/kg) took effect to a similar extent, immunological mechanisms underlying those effects were found to be distinct from each other. That is, the results obtained from several in vivo assays, including mast cell-mediated passive systemic anaphylaxis, activation/proliferation of adoptively transferred antigen-specific T cells and immunoglobulin (IgG1, IgE, IgA) production by antigen-specific B cells, illustrated that inotodiol selectively inhibited the mast cell function without having any noticeable effect on other immune responses while the crude Chaga mushroom extract indiscriminately suppressed diverse immune responses. The strong anti-allergic activity of inotodiol, along with its remarkable selectivity to mast cell, makes it an excellent therapeutic candidate for food allergy with both high efficacy and outstanding safety.

Cytotoxic triterpene saponins from the mangrove Aegiceras corniculatum.

Using various chromatographic separations, sixteen compounds, including one new triterpene saponin named aegicoroside A (1), were isolated from the leaves of the Vietnamese mangrove Aegiceras corniculatum. Their structures were determined by spectroscopic methods such as 1D and 2D NMR and HR-ESI-MS. The cytotoxic activities of the isolated compounds against MCF7 (breast), HCT116 (colon), B16F10 (melanoma), and A549 (adenocarcinoma) cancer cell lines were also evaluated. Strong cytotoxicity was observed for sakurasosaponin (2) against all four cancer cell lines and for sakurasosaponin methyl ester (3) against MCF7, A549, and HCT116 cell lines with IC50 values ranging from 2.89 ± 0.02 to 9.86 ± 0.21 µM.

Improved Transfection Efficiency and Metabolic Activity in Human Embryonic Stem Cell Using Non-Enzymatic Method.

Human embryonic stem cells (hESCs) are pluripotent cells widely used in conventional and regenerative medicine due to their ability to self-renew, proliferate and differentiate. Recently, genetic modification of stem cells using genome editing is the most advanced technique for treating hereditary diseases. Nevertheless, the low transfection efficiency of hESCs using enzymatic methods is still limited in in vitro preclinical research. To overcome these limitations, we have developed transfection methods using non-enzymatic treatments on hESCs. In this study, hESCs were transfected following enzymatic (TrypLE and trypsin) and non-enzymatic treatment ethylenediaminetetraacetic acid (EDTA) to increase transfection efficiency. Flow cytometric analysis using an enhanced green fluorescent protein vector showed a significantly increased transfection efficiency of EDTA method compared to standard enzyme method. In addition, the EDTA approach maintained stable cell viability and recovery rate of hESCs after transfection. Also, metabolic activity by using Extracellular Flux Analyzer revealed that EDTA method maintained as similar levels of cell functionality as normal group comparing with enzymatic groups. These results suggest that transfection using EDTA is a more efficient and safe substitute for transfection than the use of standard enzymatic methods.

Anti-allergic effects of the ethanol extract of Syzygium formosum (Wall.) Masam leaves and its immunoregulatory mechanisms.

ETHNOPHARMACOLOGICAL RELEVANCE: As documented in a Vietnamese traditional medical encyclopedia, Syzygium formosum (Wall.) Masam leaves have been routinely used among indigenous Vietnamese people for treatment of various allergy-like symptoms including dermatitis and rhinitis. AIM OF THE STUDY: Anti-allergic activity of S. formosum leaves was examined with a mouse model of chicken ovalbumin (cOVA)-induced food allergy, and mechanisms underlying the anti-allergic effect were explored. MATERIAL AND METHODS: BALB/c mice were administered i.p. cOVA (20µg) plus alum (2mg) twice on day 0 and 14 for sensitization (immunization). Two weeks after the second immunization, the mice were administered cOVA (50mg) p.o. 5 times every 3 days to induce food allergy symptoms (i.e., anaphylaxis, diarrhea, and drop in the body temperature). Ethanol extract of dried leaves of S. formosum (80mg/kg or 200mg/kg body weight) was administered p.o. daily during the induction (challenge) period. RESULTS: Treatment with the S. formosum leaves ethanol extract ameliorated the allergic symptoms to a significant extent and in a dose-dependent manner. The treatment also resulted in a significant improvement in the inflammatory lesion in the small intestine and reduction in the numbers of mast cells and eosinophils recruited to the lesion. The treatment also brought about a significant reduction in the levels of Th2 cytokines produced by the mesenteric lymph node cells cultured ex vivo with cOVA. The passive anaphylaxis experiment also showed that the extract treatment impaired the mast cell function. CONCLUSION: Our study provides a scientific basis for the traditional (indigenous) use of the S. formosum leaves extract for the treatment of various allergy symptoms in Vietnam. In addition, the results show that the extract has activities to suppress antigen-specific Th2 T cell immune responses and the mast cell function, which are directly related with its anti-allergic effect.

A new rearranged abietane diterpene from Clerodendrum inerme with antioxidant and cytotoxic activities.

Eight compounds were isolated from the leaves of Clerodendrum inerme, including one new rearranged abietane diterpene, crolerodendrum B (1). Their structures were determined by means of spectroscopic methods including one-dimensional and two-dimensional nuclear magnetic resonance (1-D and 2-DNMR), high-resolution electrospray ionisation mass spectrometry (HR-ESI-MS) and circular dichroism (CD). The DPPH radical scavenging and cytotoxic activities of isolated compounds against MCF7 (breast), HCT116 (colon) and B16F10 (melanoma) cancer cell lines were evaluated. Compounds 1, 3 and 4 exhibited strong DPPH radical-scavenging effects (ED50 values of 17.6 ± 2.1, 10.1 ± 0.8 and 11.3 ± 0.3 µM, respectively) and 4 showed strong cytotoxicity against the HCT116 cell line (IC50 = 3.46 ± 0.01 µM).

The mast cell stabilizing activity of Chaga mushroom critical for its therapeutic effect on food allergy is derived from inotodiol.

While an anti-allergic effect of Chaga mushroom (Inonotus obliquus) has been indicated, its therapeutic effect on allergy and immunoregulatory mechanisms and chemical constituents directly responsible for that are hardly known. We examined the effect of 70% ethanol extract of Chaga mushroom (EE) and its dichloromethane (DF) and aqueous (AF) fractions using a mouse model of chicken ovalbumin (cOVA)-induced food allergy, and found that only EE and DF ameliorated allergy symptoms to a significant extent. The in vivo mast cell-stabilizing activity was also found only in EE and DF whereas the activities to suppress Th2 and Th17 immune responses and cOVA-specific IgE production in the small intestine were observed in all three treatment regimens, implying that inhibition of the mast cell function by lipophilic compounds was vital for the therapeutic effect. Results also indicated that inotodiol, a triterpenoid predominantly present in DF, played an active role as a mast cell stabilizer.

Potentiation of T Cell Stimulatory Activity by Chemical Fixation of a Weak Peptide-MHC Complex.

The stability of peptide-MHC complex (pMHC) is an important factor to shape the fate of peptide-specific T cell immune response, but how it influences on T cell activation process is poorly understood. To better understand that, we investigated various T cell activation events driven by Ld MHCI loaded with graded concentrations of P2Ca and QL9 peptides, respectively, with 2C TCR Tg T cells; the binding strength of P2Ca for Ld is measurably weaker than that of QL9, but either peptides in the context of Ld interact with 2C TCR with a similar strength. When their concentrations required for early T cell activation events, which occur within several minutes to an hour, were concerned, EC50s of QL9 were about 100 folds lower than those of P2Ca, which was expected from their association constants for Ld. When EC50s for late activation events, which takes over several hours to occur, were concerned, the differences grew even larger (> 300 folds), suggesting that, due to weak binding, Ld/P2Ca dissociate from each other more easily to lose its antigenicity in a short time. Accordingly, fixation of Ld/P2Ca with paraformaldehyde resulted in a significant improvement in its immunogenicity. These results imply that binding strength of a peptide for a MHC is a critical factor to determine the duration of pMHC-mediated T cell activation and thus the attainment of productive T cell activation. It is also suggested that paraformaldehyde fixation should be an effective tool to ameliorate the immunogenicity of pMHC with a poor stability.

Chemical Components from Aloe and their Inhibition of Indoleamine 2, 3-dioxygenase.

BACKGROUND: In Korea, Aloe is routinely ingested as a traditional medicine or as a component of health beverages. OBJECTIVE: To research the inhibition of indoleamine 2, 3-dioxygenase (IDO) activities of components from Aloe. MATERIALS AND METHODS: the compounds were isolated by a combination of silica gel and YMC Rp-18 column chromatography, and their structures were identified by analysis of spectroscopic data (1D, 2D-NMR, and MS). All of the isolated compounds were examined for their ability to inhibit IDO, which actively suppresses immune functions by catalyzing the rate limiting reaction in the conversion of tryptophan to kynurenine. RESULTS: In this phytochemical study, 18 known compounds were isolated from aqueous dissolved Aloe exudates. All of the isolated compounds were examined for their ability to inhibit IDO activities for a series of anthraquinone derivatives (1-7) isolated from the Aloe extract; the IC50 values of these compounds ranged from 39.41 to 53.93 µM. Enzyme kinetic studies of their modes of inhibition indicated that all of the compounds were uncompetitive inhibitors. CONCLUSION: The aqueous dissolved Aloe exudate can be used as a source of novel natural IDO inhibitors and merit testing as therapeutic agents in the treatments of cancer and immunopathologic diseases, such as autoimmune, inflammatory, and allergic disorders. SUMMARY: In this study, 18 known compounds were isolated from aqueous dissolved Aloe exudates. All of the isolated compounds were examined for their ability to inhibit indoleamine 2, 3-dioxygenase (IDO) activities for a series of anthraquinone derivatives (1-7) isolated from the Aloe extract. Abbreviation used: IDO: inhibit indoleamine 2, 3-dioxygenase, TMS: tetramethylsilane, HMQC: heteronuclear multiple quantum correlation, HMBC: heteronuclear multiple bond correlation, COSY: 1H-1H correlation spectroscopy, ESI-MS: Electrospray ionization mass spectrometry, DMSO: dimethyl sulfoxide.

GEMINI: Initial behavioral results after full severance of the cervical spinal cord in mice.

BACKGROUND: The GEMINI spinal cord fusion protocol has been developed to achieve a successful cephalosomatic anastomosis. Here, we report the preliminary data on the use of a fusogen [polyethylene glycol (PEG)] after full cervical cord transection in mice to facilitate the fusion of both ends of a sharply transected spinal cord. METHODS: Cervical laminectomy and a complete, visually confirmed cervical cord (C 5) transection was performed on female albino mice (n = 16). In Group 1 (n = 8), a fusogen, (PEG) was used to bridge the gap between the cut ends of the spinal cord. Group 2 received the same spinal cord transection but was treated with saline. Outcome was assessed daily using a standard scale (modified 22-point Basso-Beattie-Bresnahan scale) and filmed on camera. RESULTS: The PEG group (group 1) showed partial restoration of motor function after 4 weeks of observation; group 2 (placebo) did not recover any useful motor activity. CONCLUSION: In this preliminary experiment, PEG, but not saline, promoted partial motor recovery in mice submitted to full cervical transection.

Spinal cord fusion with PEG-GNRs (TexasPEG): Neurophysiological recovery in 24 hours in rats.

BACKGROUND: The GEMINI spinal cord fusion protocol has been developed to achieve a successful cephalosomatic anastomosis. Here, for the first time, we report the effects of locally applied water-soluble, conductive PEG(polyethylene glycol)ylated graphene nanoribbons (PEG-GNRs) on neurophysiologic conduction after sharp cervical cord transection in rats. PEG-GNRs were produced by the polymerization of ethylene oxide from anion-edged graphene nanoribbons. These combine the fusogenic potential of PEG with the electrical conducting properties of the graphene nanoribbons. METHODS: Laminectomy and transection of cervical spinal cord (C5) was performed on Female Sprague-Dawley (SD) rats. After applying PEG-GNR on the severed part, electrophysiological recovery of the reconstructed cervical spinal cord was confirmed by somatosensory evoked potentials (SSEPs) at 24 h after surgery. RESULTS: While no SSEPs were detected in the control group, PEG-GNR treated group showed fast recovery of SSEPs at 24 h after the surgery. CONCLUSION: In this preliminary dataset, for the first time, we report the effect of a novel form of PEG with the goal of rapid reconstruction of a sharply severed spinal cord.

Accelerated recovery of sensorimotor function in a dog submitted to quasi-total transection of the cervical spinal cord and treated with PEG.

BACKGROUND: A case report on observing the recovery of sensory-motor function after cervical spinal cord transection. CASE DESCRIPTION: Laminectomy and transection of cervical spinal cord (C5) was performed on a male beagle weighing 3.5 kg. After applying polyethylene glycol (PEG) on the severed part, reconstruction of cervical spinal cord was confirmed by the restoration of sensorimotor function. Tetraplegia was observed immediately after operation, however, the dog showed stable respiration and survival without any complication. The dog showed fast recovery after 1 week, and recovered approximately 90% of normal sensorimotor function 3 weeks after the operation, although urinary disorder was still present. All recovery stages were recorded by video camera twice a week for behavioral analysis. CONCLUSION: While current belief holds that functional recovery is impossible after a section greater than 50% at C5-6 in the canine model, this case study shows the possibility of cervical spinal cord reconstruction after near-total transection. Furthermore, this case study also confirms that PEG can truly expedite the recovery of sensorimotor function after cervical spinal cord sections in dogs.

Perivascular Progenitor Cells Derived From Human Embryonic Stem Cells Exhibit Functional Characteristics of Pericytes and Improve the Retinal Vasculature in a Rodent Model of Diabetic Retinopathy.

UNLABELLED: : Diabetic retinopathy (DR) is the leading cause of blindness in working-age people. Pericyte loss is one of the pathologic cellular events in DR, which weakens the retinal microvessels. Damage to the microvascular networks is irreversible and permanent; thus further progression of DR is inevitable. In this study, we hypothesize that multipotent perivascular progenitor cells derived from human embryonic stem cells (hESC-PVPCs) improve the damaged retinal vasculature in the streptozotocin-induced diabetic rodent models. We describe a highly efficient and feasible protocol to derive such cells with a natural selection method without cell-sorting processes. As a cellular model of pericytes, hESC-PVPCs exhibited marker expressions such as CD140B, CD146, NG2, and functional characteristics of pericytes. Following a single intravitreal injection into diabetic Brown Norway rats, we demonstrate that the cells localized alongside typical perivascular regions of the retinal vasculature and stabilized the blood-retinal barrier breakdown. Findings in this study highlight a therapeutic potential of hESC-PVPCs in DR by mimicking the role of pericytes in vascular stabilization. SIGNIFICANCE: This study provides a simple and feasible method to generate perivascular progenitor cells from human embryonic stem cells. These cells share functional characteristics with pericytes, which are irreversibly lost at the onset of diabetic retinopathy. Animal studies demonstrated that replenishing the damaged pericytes with perivascular progenitor cells could restore retinal vascular integrity and prevent fluid leakage. This provides promising and compelling evidence that perivascular progenitor cells can be used as a novel therapeutic agent to treat diabetic retinopathy patients.

Triterpenoids isolated from the rhizomes and roots of Gentiana scabra and their inhibition of indoleamine 2,3-dioxygenase.

Gentiana scabra Bunge (Gentianaceae) is an important traditional Chinese medicine commonly used as a stomachic or appetite stimulant. In this study, 21 triterpenoids (1-21) were isolated from a methanol extract of the rhizomes and roots of G. scabra. Their structures were elucidated by comparing spectroscopic data with reported values. Among the isolated triterpenoids, scabanol (2) was firstly isolated from natural sources. All isolated compounds were evaluated for their inhibitory activity against indoleamine 2,3-dioxygenase (IDO), which catalyzes the rate limiting reaction for the conversion of tryptophan to kynurenine. Compounds 10 and 11 showed significant inhibitory activities, with IC(50) values of 12.5 and 9.5 µM, respectively. Compound 12 showed a moderate inhibitory effect, with an IC(50) value of 18.7 µM. Compounds 2 and 13 showed weaker inhibitory effects, with IC(50) values of 56.8 and 60.6 µM, respectively. Kynurenine is a potent immune modulator to suppress the functions of a variety of immune cells including T cells and natural killer cells. Given that, our results that a few selected triterpenoids inhibit IDO warrant further studies on their effects on the host immune system as natural immune stimulators.

Mechanisms of tumor-induced T cell immune suppression and therapeutics to counter those effects.

The theory of tumor immune surveillance states that the host immune system has means to recognize transformed cells and kills them to prevent growth and spreading of those cells. Nevertheless, cancer cells often survive and outgrow to form a tumor mass and metastasize to other tissues or organs. During the stage of immune evasion of tumor, various changes takes place both in the tumor cells and the tumor microenvironment to divert the anti-tumor immune responses by T cells and natural killer cells. Advances in the basic science in tumor immunology have led to development of many creative strategies to overcome the immune suppression imposed during tumor progression, a few of which have been approved for the treatment of cancer patients in the clinic. In the first part of this review, mechanisms of tumor-induced T cell immune suppression resulting in immune evasion of tumors will be discussed. In the second part, emerging methods to harness the immune responses against tumors will be introduced.