A screening strategy for bioactive components of Bu-Zhong-Yi-Qi-Tang regulating spleen-qi deficiency based on "endobiotics-targets-xenobiotics" association network.

ETHNOPHARMACOLOGICAL RELEVANCE: Bu-Zhong-Yi-Qi-Tang is a famous traditional Chinese medicine formula that has been prevalent in China for over 700 years to treat spleen-qi deficiency related diseases, such as gastrointestinal and respiratory disorders. However, the bioactive components responsible for regulating spleen-qi deficiency remain unclear and have puzzled many researchers. AIM OF THE STUDY: The current study focuses on efficacy evaluation of regulating spleen-qi deficiency and screening the bioactive components of Bu-Zhong-Yi-Qi-Tang. MATERIALS AND METHODS: The effects of Bu-Zhong-Yi-Qi-Tang were evaluated through blood routine examination, immune organ index, and biochemical analysis. Metabolomics was utilized to analyze the potential endogenous biomarkers (endobiotics) in the plasma, and the prototypes (xenobiotics) of Bu-Zhong-Yi-Qi-Tang in the bio-samples were characterized using ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry. Then, these endobiotics were used as "bait" to predict targets based on network pharmacology and to screen potential bioactive components from the absorbed prototypes in the plasma by constructing an "endobiotics-targets-xenobiotics" association network. Further, the anti-inflammatory activities of representative compounds (calycosin and nobiletin) were validated through poly(I:C)-induced pulmonary inflammation mice model. RESULTS: Bu-Zhong-Yi-Qi-Tang exhibited immunomodulatory and anti-inflammatory activities in spleen-qi deficiency rat, as supported by the observation of increased levels of D-xylose and gastrin in serum, an increase in the thymus index and number of lymphocytes in blood, as well as a reduction in the level of IL-6 in bronchoalveolar lavage fluid. Furthermore, plasma metabolomic analysis revealed a total of 36 Bu-Zhong-Yi-Qi-Tang related endobiotics, which were mainly enriched in primary bile acids biosynthesis, the metabolism of linoleic acid, and the metabolism of phenylalanine pathways. Meanwhile, 95 xenobiotics were characterized in plasma, urine, small intestinal contents, and tissues of spleen-qi deficiency rat after Bu-Zhong-Yi-Qi-Tang treatment. Using an integrated association network, six potential bioactive components of Bu-Zhong-Yi-Qi-Tang were screened. Among them, calycosin was found to significantly reduce the levels of IL-6 and TNF-α in the bronchoalveolar lavage fluid, increase the number of lymphocytes, while nobiletin dramatically decreased the levels of CXCL10, TNF-α, GM-CSF, and IL-6. CONCLUSION: Our study proposed an available strategy for screening bioactive components of BYZQT regulating spleen-qi deficiency based on "endobiotics-targets-xenobiotics" association network.

Integrating network pharmacology and experimental verification to decipher the immunomodulatory effect of Bu-Zhong-Yi-Qi-Tang against poly (I:C)-induced pulmonary inflammation.

Pulmonary inflammation caused by respiratory tract viral infections is usually associated with acute exacerbation of respiratory diseases, such as asthma and chronic obstructive pulmonary disease (COPD). Therefore, maintaining the pulmonary immune homeostasis is particular important for prevention of the acute exacerbation. Bu-Zhong-Yi-Qi-Tang (BZYQT), a traditional Chinese medicine formula, has been broadly used to improve respiratory and gastrointestinal disorders in China for over 700 years. Previously, we have found the regulatory activity of BZYQT on the lower respiratory immune system, while its potential effects during pulmonary inflammation remain unknown. Thus, the current study focused on deciphering its immunomodulatory effect and potential mechanism against pulmonary inflammation by using a viral RNA analogue, poly (I:C), induced murine pulmonary inflammation model and BEAS-2B cell model coupled with network pharmacology. Inflammatory cells in the bronchoalveolar lavage fluid were counted through microscope examination according to the cell's morphology and staining characteristics; protein and gene levels of inflammatory mediators were determined with Elisa and quantitative PCR, respectively; network pharmacology was conducted based on 46 BZYQT-related potential bioactive components, pulmonary inflammation and immune-related targets. Our results indicated that the recruitment of neutrophils and the expression of Adgre1 (encoding the F4/80, which is a macrophage marker) in the lung induced by poly (I:C) were significantly reduced after BZYQT treatment, and these effects were further demonstrated to be related to the interference of leukocyte transendothelial migration from the decreased levels of CXCL10, IL-6, TNF-α, CXCL2, ICAM-1, VCAM-1, and E/P-selectins. Furthermore, BZYQT inhibited the CXCL10, TNF-α, and IFN-ß expression of poly (I:C)-challenged BEAS-2B cells in a dose-dependent manner. Through integrating results from network pharmacology, experiments, and the published literature, isoliquiritigenin, Z-ligustilide, atractylenolide I, atractylenolide III, formononetin, ferulic acid, hesperidin, and cimigenoside were presumed as the bioactive components of BZYQT against pulmonary inflammation. Overall, our findings demonstrated that BZYQT possesses a pronounced immunomodulatory effect on poly (I:C)-induced pulmonary inflammation, which provides a pharmacological basis for BZYQT in the treatment of respiratory disorders.

Preparation of Tenuifolin from Polygala senega L. Root Using a Hydrolytic Continuous Flow System under High-Temperature, High-Pressure Conditions.

An improved process for preparing tenuifolin (presenegenin 3-ß-d-glucopyranoside) from the root of Polygala senega L. was developed. A crude saponin mixture extracted from P. senega was subjected to hydrolysis, and the reactivity of compounds in the extract was controlled by utilizing the combination of a flow reactor and experimental design. In addition, column chromatography with HP 20, a synthetic polystyrenic adsorbent, allowed the gram-scale preparation of tenuifolin in a continuous manner with fewer steps. This approach shortens the total time required for gram-scale preparation from 16 to 5 h in a continuous manner while improving the yield from 0.59% to 2.08% (w/w).

Chemical profile of Cimicifuga heracleifolia Kom. And immunomodulatory effect of its representative bioavailable component, cimigenoside on Poly(I:C)-induced airway inflammation.

ETHNOPHARMACOLOGICAL RELEVANCE: The dried rhizome of Cimicifuga heracleifolia Kom. (C. heracleifolia) is a popular traditional Chinese medicine, which has been extensively used in Asian countries for its anti-inflammatory, antipyretic and analgesic activities. However, further utilization and application of C. heracleifolia have been hampered due to a lack of full understanding of its active ingredients. AIM OF STUDY: The present study aims for clarification of the systematical chemical profile of C. heracleifolia and the immunomodulatory effect of its main bioavailable component. MATERIALS AND METHODS: Comprehensive chemical profile of C. heracleifolia was systematically analyzed by ultra-performance liquid chromatography hyphenated with quadrupole time-of-flight tandem mass spectrometry (UPLC-Q-TOF/MS). Xenobiotics after oral administration of C. heracleifolia extracts were investigated to hunt for bioavailable components. The immunomodulatory activity evaluation of cimigenoside was achieved on poly(I:C)-induced airway inflammation mouse and BEAS-2B cell models from aspects of neutrophil infiltration, lung inflammation by using microscope analysis, quantification of production and expression of inflammatory cytokine and chemokines by using ELISA and quantitative PCR. RESULTS: By UPLC-Q-TOF/MS analysis, 110 compounds (including 81 triterpenoids, 21 cinnamic acid derivatives, and 8 other structure types) were identified or tentatively characterized in ethanolic extract of C. heracleifolia. Based on the data of chemical profile, xenobiotics of C. heracleifolia were subsequently analyzed, and triterpene glycosides were detected as the major bioavailable ingredients. Oral administration of cimigenoside, a representative triterpene glycoside, could prevent neutrophils infiltration in the lung due to suppression of the production of CXCL2 and CXCL10, and the expression of P-selectin, VCAM1 in poly(I:C)-induced airway inflammation model mice. Moreover, cimigenoside also inhibited the productions of inflammatory cytokines and chemokines from human airway epithelial cell line (BEAS-2B cells) induced by poly(I:C). CONCLUSION: Triterpene glycosides were the main components of C. heracleifolia extract, and cimigenoside was considered as the effective component with immunomodulatory effect on the pulmonary immune system by oral administration.

Antimalarial alkaloid from Hypoestes forskaolii.

Following on from previous studies, we brought further our quest for anti-malarial agents isolated from plants grown in the Saudi Arabian Peninsula. Methanolic extracts were prepared from eighteen Saudi plants and then tested in vitro to assess their anti-malarial effects on Plasmodium falciparum K1, (a chloroquine-resistant strain) as well as their cytotoxicity on MRC5 (human diploid embryonic lung cell line) cells. Moderate anti-malarial activity was observed in extracts prepared from Hypoestes forskaolii (Vahl) R. Br. (IC50 value of 5.5 µg/ml) and Rhus retinorrhaea (IC50: 7.71 µg/ml). The remaining sixteen plant extracts appeared to be inactive (IC50 > 12.5 µg/ml). A novel phenanthro-quinolizidine alkaloid, 15ß-hydroxycryptopleurine-N-oxide, was isolated from H. forskaolii using bio-guided fractionation procedures. Chloroquine-resistant (K1) and chloroquine-sensitive (FCR3) strains of P. falciparum appeared very sensitive to the anti-malarial activity of 15ß-hydroxycryptopleurine-N-oxide, giving IC50 of 6.11 and 5.13 nM respectively. It showed cytotoxicity against MRC5 "IC50 of 24.45 nM" with selectivity indices of 4.0 and 4.76 against K1 and FCR3 strains, respectively. It is our understanding that this is the first account on phenanthro-quinolizidine alkaloids anti-malarial activity on a chloroquine-resistant P. falciparum strain.

In vivo metabolic profiles of Bu-Zhong-Yi-Qi-Tang, a famous traditional Chinese medicine prescription, in rats by ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry.

Bu-Zhong-Yi-Qi-Tang (BZYQT), a famous traditional Chinese medicine prescription (TCMP), has been extensively used for conditioning sub-health status and diseases caused by spleen-qi deficiency in China for over 700 years. BZYQT is prevalent not only in China, but also in Japan and South Korea for the clinical treatment of chronic diseases, such as fatigue, tuberculosis and loss of appetite after surgery. However, due to a lack of research on the holistic metabolism of BZYQT, the in vivo bioactive components of BZYQT remain unclear, hindering further study of its in vivo mechanism of action and quality control. In the present study, a four-step integrated strategy based on ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UHPLC-Q-TOF/MS) was established to systematically screen the in vivo xenobiotics of BZYQT. Ultimately, a total of 162 xenobiotics (59 prototypes and 103 metabolites) were identified or tentatively characterized, including 48 in plasma, 147 in urine and 58 in feces, while the in vivo metabolic profile of atractylenolide III (a major component of BZYQT) was elucidated for the first time. The xenobiotics of BZYQT mainly included flavonoids from Astragali Radix, Glycyrrhizae Radix et Rhizoma and Citrus reticulatae Pericarpium; lactones from Angelicae Sinensis Radix and Atractylodis Macrocephalae Rhizoma; and triterpenoid saponins from Cimicifugae Rhizoma. After oral administration, BZYQT-related components underwent diverse metabolic pathways. Among them, flavonoids mainly underwent glucuronidation, sulfation and demethylation, while lactones mainly underwent hydroxylation and acetylcysteine conjugation, and deglycosylation was the major metabolic reaction of saponins. Our investigation gives a comprehensive analysis of the metabolic characteristics of BZYQT and will provide an important basis for further studying the pharmacokinetics of BZYQT to explore its in vivo disposal features and discover its in vivo bioactive components.

Mucosal immunomodulatory evaluation and chemical profile elucidation of a classical traditional Chinese formula, Bu-Zhong-Yi-Qi-Tang.

ETHNOPHARMACOLOGICAL EVIDENCE: With fast development and high pace life in modern society, autoimmune diseases like inflammatory bowel disease had become increasingly common. Bu-Zhong-Yi-Qi-Tang (BZYQT), a famous traditional Chinese medicine prescription (TCMP), has been used for 700 years mainly in Eastern Asia countries for the treatment of gastrointestinal and respiratory disorder, and weakness after serious diseases. These diseases were proved to be closely related to human immune system, among which, mucosal immune system is the largest immune system. So it is necessary to discover the mucosal immune related bioactive components of BZYQT. AIM OF THE STUDY: To evaluate the mucosal immunomodulatory bioactivity of BZYQT and ingredients. MATERIALS AND METHODS: Peyer's patches were collected from mice administrated orally with BZYQT, its related Octadecylsilane (ODS) fractions and polysaccharide part. Productions of several cytokines including IL-2, IL-4, IL-5, and IFN-γ from T lymphocytes were tested with enzyme linked immunosorbent assay (ELISA) by Peyer's patch cells ex vivo experiments. Chemical profile including low molecular part and polysaccharide part were investigated. Low molecular part of BZYQT and related ODS fractions were analyzed by ultra-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UPLC-Q/TOF-MS) based on LC-MS information from self-established compound library. exclusion chromatography, and chemical property has been analyzed. RESULT: Three-days' administration of BZYQT enhanced productions of IL-4 and IFN-γ in T lymphocytes of Peyer's patches in addition to IL-2. Some hydrophobic low molecular weight fractions (30%, 70% and 100% MeOH ODS fraction), which were fractionated from BZYQT by ODS column chromatography, showed enhancing or suppressive effects on productions of IL-2, IL-4 or IL-5 in T lymphocytes of Peyer's patches after oral administration. Besides, 161 components from hydrophobic low molecular weight fractions of BZYQT were unequivocally identified or tentatively characterized by UPLC-Q/TOF-MS according to retention time behaviors and fragments, and most of them were flavonoids and saponins from Glycyrrhizae Radix, Citri Reticulatae Pericarpium, and Cimicifugae Rhizoma. Polysaccharide part was separated and purified both by anion-exchange and size. BZYQT also contained at least one neutral and three weakly or strongly acidic polysaccharides, and analysis of their chemical properties indicated that a neutral polysaccharide was glucan, and acidic polysaccharides possessed heteroglycan and pectic arabinogalactan features. Murine administration of polysaccharide fractions of BZYQT induced different changes on functions of T lymphocytes in Peyer's patches from hydrophobic low molecular weight fractions. By experiment using intranasally-immunized mice, BZYQT negatively regulated antibody response in lung as combinatorial actions of its low molecular weight ingredients and polysaccharides. CONCLUSION: BZYQT contains several low and macromolecular weight ingredients, which affect to immune-function of T lymphocytes in Peyer's patches, and the formula expresses its regulative activity on lower respiratory immune system through combinatorial actions of these ingredients on immunocompetent cells in Peyer's patches.

Peyer's patch-immunomodulating glucans from sugar cane enhance protective immunity through stimulation of the hemopoietic system.

The aim of the present study was chemical clarification of in vitro Peyer's patch-immunomodulating polysaccharides in sugar cane molasses, and evaluation of in vivo modulating activity on immune function of T lymphocytes in Peyer's patches and on microenvironment of hemopoietic system. Five kinds of glucans, comprising of dextranase-sensitive and activity-related d-glucosyl moieties, were purified as in vitro Peyer's patch-immunomodulating polysaccharides from the molasses. Oral administration of a glucan-enriched subfraction induced IL-2 and GM-CSF-producing T lymphocytes in Peyer's patches, resulting in enhancement of IL-6 production in a hemopoietic microenvironment to boost neutrophil numbers in the peripheral blood stream. Oral administration of purified glucan or glucan-enrich sub-fraction of sugar cane reduced the number of Plasmodium berghei- or P. yoelii-infected erythrocytes in a murine infection model, using polysaccharide alone or via co-administration with the antimalarial drug, artesunate. These results suggested that Peyer's patch-immunomodulating glucans enhanced protective immunity through axis of Peyer's patches-hemopoietic system.

Tilia tomentosa pectins exhibit dual mode of action on phagocytes as ß-glucuronic acid monomers are abundant in their rhamnogalacturonans I.

Silver linden flowers contain different pectins (PSI-PSIII) with immunomodulating properties. PSI is a low-esterified pectic polysaccharide with predominant homogalacturonan region, followed by rhamnogalacturonan I (RGI) with arabinogalactan II and RGII (traces) domains. PSII and PSIII are unusual glucuronidated RGI polymers. PSIII is a unique high molecular weight RGI, having almost completely O-3 glucuronidated GalA units with >30% O-3 acetylation at the Rha units. Linden pectins induced reactive oxygen species (ROS) and NO generation from non-stimulated whole blood phagocytes and macrophages, resp., but suppressed OZP-(opsonized zymosan particles)-activated ROS generation, LPS-induced iNOS expression and NO production. This dual mode of action suggests their anti-inflammatory activity, which is known for silver linden extracts. PSI expressed the highest complement fixation and macrophage-stimulating activities and was active on intestinal Peyer's patch cells. PSIII was active on non-stimulated neutrophils, as it induced ß2-integrin expression, revealing that acetylated and highly glucuronidated RGI exhibits immunomodulating properties via phagocytes.

The common lavender (Lavandula angustifolia Mill.) pectic polysaccharides modulate phagocytic leukocytes and intestinal Peyer's patch cells.

Two pectic (chPS-L1, chPS-L2) and one polyphenolic (chPP-L) fractions were obtained from lavender flowers after boiling water extraction, exhaustive removing of alcohol-soluble molecules and SEC. chPS-L1 (52.4kDa) contains mainly low-acetylated and high-methoxylated homogalacturonans (HG), and smaller rhamnogalacturonan (RG) I backbone fragments rich in 1,3,5-branched arabinan and arabinogalactan (AG) II side chains. chPS-L2 (21.8kDa) contains predominantly similarly esterified HG, followed by RGI with AGII structures and RGII. The prevalence of catechin and epicatechin in chPP-L indicates that they form weak interactions with pectins. chPS-L1 and chPS-L2 enhanced ß2-integrin expression on neutrophils, inducing ROS generation and macrophage NO production. Both the effects on ß2-integrin and high complement fixation activity of chPS-L1 were proposed for its inhibitory action against PMA- and OZP-activated ROS formation. This, together with suppression of NO generation after co-stimulation with chPS-L1 and LPS, suggested anti-inflammatory activity of studied pectins. Lavender polysaccharides expressed intestinal Peyer's patch immunomodulating activity.

Acidic polysaccharide complexes from purslane, silver linden and lavender stimulate Peyer's patch immune cells through innate and adaptive mechanisms.

Three polysaccharide complexes (PSCs) were isolated from the aerial parts of common purslane (Portulaca oleracea L.), and the flowers of common lavender (Lavandula angustifolia Mill.) and silver linden (Tilia tomentosa Moench) by boiling water extraction and ethanol precipitation. The chemical composition and immunomodulating effects of isolated PSCs were characterized. The chemical characterization revealed that the three samples contain mainly pectic polysaccharides. They exhibited ex vivo intestinal immunomodulating activity through the murine Peyer's patch-mediated bone marrow cell proliferation test at 100µg/ml concentration. At the same time, they stimulated ex vivo human blood T-cell populations (CD4+/CD25+ and CD8+/CD25+), phagocytic leukocytes (CD14+ and CD64+ cells) and induced IL-6 production from human white blood cells and Peyer's patch cells. The herbal PSCs stimulated ex vivo ROS production from whole blood phagocytes and showed unspecific in vitro anti-proliferative activity against normal and A549, HeLa and LS180 tumor cells. This is the first report on immunomodulating studies of linden flower pectins and chemical and biological activity characterization of lavender polysaccharides. Our study demonstrates that similarly to purslane, lavender and silver linden herbal materials contain immunomodulating polysaccharides that could be useful for support of compromised immune system.

Synthesis of Bisdesmosidic Oleanolic Acid Saponins via a Glycosylation-Deprotection Sequence under Continuous Microfluidic/Batch Conditions.

We report the first synthesis of a series of bisdesmosidic oleanolic acid saponins using microflow reactor Comet X-01 via a continuous flow glycosylation-batch deprotection sequence. The main results of this study can be summarized as follows: (1) The microfluidic glycosylation of oleanolic acid at C-28 was achieved in quantitative yield and was applied to the synthesis of six C-28-monoglycosidic saponins. (2) The microfluidic glycosylation of oleanolic acid at C-3 was achieved in good yield without orthoester byproduct formation and was applied to the synthesis of three bisdesmosidic saponins. (3) The continuous synthesis of saponins via a microfluidic glycosylation-batch deprotection sequence was achieved in four steps involving two purifications. Thus, the continuous microfluidic glycosylation-deprotection process is expected to be suitable for the preparation of a library of bisdesmosidic oleanolic acid saponins for in vivo pharmacological studies.

Kososan, a Kampo medicine, prevents a social avoidance behavior and attenuates neuroinflammation in socially defeated mice.

BACKGROUND: Kososan, a Kampo (traditional Japanese herbal) medicine, has been used for the therapy of depressive mood in humans. However, evidence for the antidepressant efficacy of kososan and potential mechanisms are lacking. Recently, it has been recognized that stress triggers neuroinflammation and suppresses adult neurogenesis, leading to depression and anxiety. Here, we examined whether kososan extract affected social behavior in mice exposed to chronic social defeat stress (CSDS), an animal model of prolonged psychosocial stress, and neuroinflammation induced by CSDS. METHODS: In the CSDS paradigm, C57BL/6J mice were exposed to 10 min of social defeat stress from an aggressive CD-1 mouse for 10 consecutive days (days 1-10). Kososan extract (1.0 g/kg) was administered orally once daily for 12 days (days 1-12). On day 11, the social avoidance test was performed to examine depressive- and anxious-like behaviors. To characterize the impacts of kososan on neuroinflammation and adult neurogenesis, immunochemical analyses and ex vivo microglial stimulation assay with lipopolysaccharide (LPS) were performed on days 13-15. RESULTS: Oral administration of kososan extract alleviated social avoidance, depression- and anxiety-like behaviors, caused by CSDS exposure. CSDS exposure resulted in neuroinflammation, as indicated by the increased accumulation of microglia, the resident immune cells of the brain, and their activation in the hippocampus, which was reversed to normal levels by treatment with kososan extract. Additionally, in ex vivo studies, CSDS exposure potentiated the microglial pro-inflammatory response to a subsequent LPS challenge, an effect that was also blunted by kososan extract treatment. Indeed, the modulatory effect of kososan extract on neuroinflammation appears to be due to a hippocampal increase in an anti-inflammatory phenotype of microglia while sparing an increased pro-inflammatory phenotype of microglia caused by CSDS. Moreover, reduced adult hippocampal neurogenesis in defeated mice was recovered by kososan extract treatment. CONCLUSIONS: Our findings suggest that kososan extract prevents a social avoidant behavior in socially defeated mice that is partially mediated by the downregulation of hippocampal neuroinflammation, presumably by the relative increased anti-inflammatory microglia and regulation of adult hippocampal neurogenesis. Our present study also provides novel evidence for the beneficial effects of kososan on depression/anxiety and the possible underlying mechanisms.

Syntheses and mucosal adjuvant activity of simplified oleanolic acid saponins possessing cinnamoyl ester.

A series of new simplified oleanolic acid saponins with a glycosyl ester moiety at C28, were efficiently prepared. Furthermore, the effect of nasal administration of the synthetic oleanolic acid saponins on the nasal anti-influenza virus antibody titer against secondary nasal inoculation of the influenza split vaccine was examined. The result revealed cinnamoyl saponin as a suitable candidate vaccine adjuvant.

Alleviative Effects of a Kampo (a Japanese Herbal) Medicine "Maoto (Ma-Huang-Tang)" on the Early Phase of Influenza Virus Infection and Its Possible Mode of Action.

A Kampo medicine, maoto, has been prescribed in an early phase of influenza-like illness and used for a treatment of influenza clinically in Japan these days. However, the efficacy of maoto against the virus infection remains to be elucidated. This study was conducted to evaluate the alleviative effects of maoto against early phase of influenza virus infection and its preliminary mode of actions through immune systems. When maoto (0.9 and 1.6 g/kg/day) was orally administered to A/J mice on upper respiratory tract infection of influenza virus A/PR/8/34 from 4 hours to 52 hours postinfection (p.i.) significant antipyretic effect was shown in comparison with water-treated control. Administration of maoto (0.8 and 1.3 g/kg/day) significantly decreased the virus titers in both nasal (NLF) and bronchoalveolar lavage fluids (BALF) at 52 hours p.i., and significantly increased the anti-influenza virus IgM, IgA, and IgG1 antibody titers in NLF, BALF, and serum, respectively. Maoto also increased significantly the influenza virus-bound IgG1 and IgM antibody titers in serum and the virus-bound IgM antibody titer in even the BALF of uninfected A/J mice. These results indicate that maoto exerts antipyretic activity in influenza virus-infected mice and virus reducing effect at an early phase of the infection through probably augmentation of the virus-bound natural antibodies.

Intestinal Peyer's patch-immunomodulating glucomannans from rhizomes of Anemarrhena asphodeloides Bunge.

During screening for intestinal Peyer's patch-immunomodulating polysaccharides from plant resources including medicinal herbs, a potent modulating activity was observed in a crude polysaccharide fraction (AS-1) from the rhizome of Anemarrhena asphodeloides Bunge. Oral administration of AS-1 (100 mg/kg/day) to aged BALB/c mice enhanced productions of IL-10, IFN-γ and IL-6 from Peyer's patch immunocompetent cells, and its oral administration to ovalbumin (OVA)-fed B10.A mice led to significant suppression on induction of OVA-specific IgE in systemic immune system. Further fractionation of the polysaccharides in the crude polysaccharide fraction, AS-1, yielded 4 polysaccharide fractions that were potently active, and contained glucomannans. Treatment of these polysaccharide fractions with endo-ß-D-(1→4)-mannanase significantly decreased their activities. Mannanase digestion of the active glucomannan gave both long and short hexosyl-oligosaccharides, whereas konjac glucomannan, which was inactive, released short oligosaccharides. Structural analysis indicates that the long oligosaccharides from the active glucomannan contain mannanase-resistant complex structure comprising ß-D-Man and ß-D-Glc.

Immunomodulating pectic polysaccharides from waste rose petals of Rosa damascena Mill.

A water-soluble polysaccharide (RP-1) was obtained from distilled rose petals of Rosa damascena Mill. as an attempt for valorization of the waste. RP-1 showed in vitro intestinal immune system modulating activity through Peyer's patch cells and IL-6 producing activity from macrophages. RP-1 lost most of its immunomodulating activity by degradation of the carbohydrate moiety with periodate. RP-1 was fractionated by anion-exchange and gel filtration chromatography and some of the fractions showed significant intestinal immune system modulating activity. The active fractions were suggested to be pectic polysaccharides and type II arabino-3,6-galactan from the component sugar analyses and the reactivity with Yariv antigen. When some active fractions were digested with endo α-d-(1→4)-polygalacturonase, highest molecular weight fragments which were considered as rhamnogalacturonan I, showed potent immunomodulating activities. To our knowledge, this is a first report which explores the possibility for utilization of waste rose petals as a source of immunomodulating pectic polysaccharides.

Modulation of chemokine expression on intestinal epithelial cells by Kampo (traditional Japanese herbal) medicine, Hochuekkito, and its active ingredients.

The intestinal epithelial cells sit at the interface between a lumen and a lamina propria or lymph nodes such as Peyer's patches, where they play important roles in maintaining intestinal homeostasis through chemokine secretion. This study investigated the effect of Hochuekkito (TJ-41)-a traditional Japanese herbal (Kampo) formula used as a tonic for weakness-on chemokine expression in intestinal epithelial cells in order to explore the mechanism of its modulating effect against mucosal immunity. When cells from the rat normal small intestinal epithelial cell-line IEC-6 were stimulated with TJ-41, mRNA expression of CC chemokine ligand (CCL) 11 (eotaxin), CCL20 (MIP-3α) and CCL25 (TECK) was enhanced. Oral administration of TJ-41 to methotrexate-treated mice enhanced mRNA expression of CCL25 and keratinocyte growth factor in the jejunum with, decreasing mRNA expression of the inflammatory marker tumor necrosis factor (TNF)-α. Although oral administration of TJ-41 did not affect CCL20 mRNA expression in villus epithelium of methotrexate-treated mice, enhancement of CCL20 mRNA expression was observed in Peyer's patches. Immunohistochemical analysis detected dense staining with anti-CCL20 antibody in the follicle-associated epithelium region of Peyer's patches in mice administered TJ-41. Analysis of active ingredients indicates that polysaccharide-containing macromolecules in TJ-41 contribute to the enhancement of CCL20 mRNA expression through an intracellular signal cascade via nuclear factor kappa B (NF-κB) activation.

In vitro anti-influenza virus activity of a cardiotonic glycoside from Adenium obesum (Forssk.).

Methanolic extracts of six Saudi plants were screened for their in vitro antiviral activity using influenza virus A/PR/8/34 (H1N1) and MDCK cells in an MTT assay. The results indicated that the extracts of Adeniumobesum and Tephorosianubica possessed antiviral activity (99.3 and 93.3% inhibition at the concentration of 10 µg/ml, respectively). Based on these results A. obesum was selected for further study by applying bioactivity-guided fractionation to isolate its antiviral principle. The antiviral principle was isolated from the chloroform fraction through solvent fractionation, combined open liquid chromatography and HPLC. The isolated active compound A was identified as oleandrigenin-ß-D-glucosyl (1→4)-ß-D-digitalose, on the basis of its spectral analysis (MS, 1D and 2D NMR). The isolated glycoside showed reduction of virus titre by 69.3% inhibition at concentration of 1 µg/ml (IC(50)=0.86 µg/ml).