Molecular Mechanism behind the Safe Immunostimulatory Effect of Withania somnifera.

Withania somnifera (L.) Dunal (family Solanaceae) is a medicinal plant known for, among many pharmacological properties, an immune boosting effect. Our recent study revealed that its key immunostimulatory factor is lipopolysaccharide of plant-associated bacteria. This is peculiar, because, although LPS can elicit protective immunity, it is an extremely potent pro-inflammatory toxin (endotoxin). However, W. somnifera is not associated with such toxicity. In fact, despite the presence of LPS, it does not trigger massive inflammatory responses in macrophages. To gain insights into the safe immunostimulatory effect of W. somnifera, we conducted a mechanistic study on its major phytochemical constituent, withaferin A, which is known for anti-inflammatory activity. Endotoxin-triggered immunological responses in the presence and absence of withaferin A were characterized by both in vitro macrophage-based assay and in vivo cytokine profiling in mice. Collectively, our results demonstrate that withaferin A selectively attenuates the pro-inflammatory signaling triggered by endotoxin without impairing other immunological pathways. This finding provides a new conceptual framework to understand the safe immune-boosting effect of W. somnifera and possibly other medicinal plants. Furthermore, the finding opens a new opportunity to facilitate the development of safe immunotherapeutic agents, such as vaccine adjuvants.

Pistachio shells as remediating agents for uranium in contaminated industrial seawater.

Waterways have histories of being contaminated by heavy and/or radioactive metals produced by industrial processes. Natural radioisotopes of uranium (238U, 235U and 234U), long-lived radiometals, are widespread in the environment as a result of both naturally occurring processes and anthropogenic processes. Uranium is considered a major threat to humans. Previous research has focused on using inorganic materials (e.g. ion-exchangers, extractants, nanoporous sorbents) to remove such metal. However, there has been a rise in using biodegradable, recyclable, and organic biological wastes to remove heavy toxic metals from aqueous solutions. The purpose of this study is to identify pistachio shells as good candidates for the removal of uranyl from aqueous solutions. The influences of pH, contact time, temperature, and initial uranyl concentration on uranyl uptake were investigated. The influence of pH was observed to be variable, with relatively high uptake occurring at pH 4 and at slightly alkaline pH values. Uptake increased as a function of contact time, temperature, and initial uranyl concentration. The mechanism followed pseudo-second-order and intraparticle kinetics models, and the shell was demonstrated to be a Freundlich isotherm. The shells were successfully demonstrated to be viable adsorbents for uranium in seawater samples, with obtained trends similar to those achieved in the batch studies.

Cell-line-based assay for the toxicity/benefit analysis of lipopolysaccharides in plants.

There are many immune-boosting medicinal plants that can potently activate innate immune cells. Recent studies indicate that the active factors of some immune-boosting plants are lipopolysaccharides (LPSs) of plant-associated bacteria. However, little is currently known about the potential risk and benefit of LPSs in medicinal plants. To facilitate their characterization, we established a simple cell-line-based assay that can be used to screen the toxicity and benefit of LPSs in medicinal plants. The assay can distinguish endotoxic diphosphoryl lipid A (DPL) from beneficial monophosphoryl lipid A (MPL), which is a clinically used immunological adjuvant for vaccines. The established assay was used to characterize commercial supplements of Ashwagandha, which was shown to contain immunostimulatory LPSs. The study revealed that Ashwagandha activates macrophages in a manner similar to MPL. The current finding underscores the importance of further studies to characterize the LPSs in immune-boosting medicinal plants.

Abundance of Plant-Associated Gammaproteobacteria Correlates with Immunostimulatory Activity of Angelica sinensis.

Background: Angelica sinensis is a medicinal plant known for a variety of biological effects, including its ability to stimulate innate immune cells in humans. Recent studies indicate that the immunostimulatory activity of A. sinensis arises from microbe-associated molecular patterns (MAMPs) of plant-associated bacteria. However, it is unknown which bacterial taxa in A. sinensis are responsible for the production of immunostimulatory MAMPs. Methods: Samples of A. sinensis were subjected to a cell-based assay to detect monocyte-stimulation and 16S ribosomal RNA amplicon sequencing, which revealed their immunostimulatory activity and microbial communities. The resulting data were analyzed by Linear discriminant analysis effect size (LEfSe), an online biostatistical tool for metagenomic biomarker discovery, to identify the bacterial taxonomical features correlated with the immunostimulatory activity. Results: A series of bacterial taxa under Gammaproteobacteria correlated positively with the immunostimulatory activity, whereas several Gram-positive taxa and Betaproteobacteria correlated negatively with the activity. Conclusions: The identified bacterial taxa set a new stage to characterize immunostimulatory MAMPs in plants.

Identification of ginkgolide targets in brain by photoaffinity labeling.

Ginkgolides are terpene trilactones in Ginkgo biloba, a popular medicinal herb for memory disorders. Although ginkgolides are known for various neurobiological effects, their macromolecular target in brain is unknown. In this work, we employed benzophenone derivatives of ginkgolides to identify their binding target in brain. Photolabeling of bovine hippocampus homogenates identified a series of α-tubulin isotypes. Selective photolabeling of α-tubulin over ß-tubulin, which is equally abundant in brain, suggested that ginkgolides might modulate microtubule biology differently than typical microtubule-binding agents, such as taxol. In fact, ginkgolide A did not affect microtubule polymerization or cell proliferation; instead, it inhibited detyrosination of α-tubulin and reorientation of microtubule-organizing centers. Taken together, the current findings indicate that ginkgolides constitute a new class of microtubule-binding agents with distinct effects on α-tubulin biology.

Uncovering potential 'herbal probiotics' in Juzen-taiho-to through the study of associated bacterial populations.

Juzen-taiho-to (JTT) is an immune-boosting formulation of ten medicinal herbs. It is used clinically in East Asia to boost the human immune functions. The active factors in JTT have not been clarified. But, existing evidence suggests that lipopolysaccharide (LPS)-like factors contribute to the activity. To examine this possibility, JTT was subjected to a series of analyses, including high resolution mass spectrometry, which suggested the presence of structural variants of LPS. This finding opened a possibility that JTT contains immune-boosting bacteria. As the first step to characterize the bacteria in JTT, 16S ribosomal RNA sequencing was carried out for Angelica sinensis (dried root), one of the most potent immunostimulatory herbs in JTT. The sequencing revealed a total of 519 bacteria genera in A. sinensis. The most abundant genus was Rahnella, which is widely distributed in water and plants. The abundance of Rahnella appeared to correlate with the immunostimulatory activity of A. sinensis. In conclusion, the current study provided new pieces of evidence supporting the emerging theory of bacterial contribution in immune-boosting herbs.

Biomarker-guided screening of Juzen-taiho-to, an oriental herbal formulation for immunostimulation.

Juzen-taiho-to is an immunostimulatory herbal formulation that is clinically used in East Asia for cancer patients undergoing chemotherapy and radiation. The formulation stimulates various leukocytes, including T, B, and NK cells and macrophages. Although Juzen-taiho-to is known to contain numerous compounds with various pharmacological activities, it is not clear which compounds are responsible for the stimulation of individual cell types. Here, we conducted what we call "biomarker-guided screening" to purify compounds responsible for the macrophages stimulatory activity. To this end, gene expression was analyzed by a DNA array for macrophages treated with Juzen-taiho-to and DMSO (vehicle control), which identified intercellular adhesion molecule 1 as a biomarker of macrophage stimulation by Juzen-taiho-to. A quantitative reverse transcription polymerase chain reaction assay of intercellular adhesion molecule 1 was then used to guide the purification of active compounds. The screening resulted in the purification of a glycolipid mixture, containing ß-glucosylceramides. The glycolipid mixture potently stimulated intercellular adhesion molecule 1 expression in primary dendritic cells as well as in primary CD14+ (macrophages) cells. The identification of this glycolipid mixture opens up an opportunity for further studies to understand how plant-derived glycolipids stimulate macrophages and dendritic cells in a safe and effective manner as demonstrated by Juzen-taiho-to.

Immunostimulatory lipid nanoparticles from herbal medicine.

Reproducibility is an important issue in biological characterization of drug candidates and natural products. It is not uncommon to encounter cases in which supposedly the same sample exhibits very different biological activities. During our characterization of macrophage-stimulatory lipids from herbal medicine, it was found that the potency of these lipids could vary substantially from experiment to experiment. Further analysis of this reproducibility issue led to the discovery of solvent-dependent nanoparticle formation by these lipids. While larger nanoparticles (approximately 100 nm) of these lipids showed modest macrophage-stimulatory activity, smaller nanoparticles (<10 nm) of the same lipids exhibited substantially higher potency. Thus, the study revealed an unexpected link between nanoparticle formation and macrophage-stimulatory activity of plant lipids. Although nanoparticles have been extensively studied in the context of vehicles for drug delivery, our finding indicates that drugs themselves can form nanoassemblies, and their biological properties may be altered by the way they assemble.

Three hydroxylated lupeol-based triterpenoid esters isolated from the Eastern Nigeria mistletoe parasitic on Kola acuminata.

Column fractionation and purification of an n-hexane fraction led to the isolation of three lupeol-based triterpenoid esters from the leaves of the Eastern Nigeria mistletoe, Loranthus micranthus Linn parasitic on a local kola nut tree, Kola acuminata. These three compounds were adequately characterised using a combination of UV/visible, IR, NMR (¹³C-NMR and ¹H-NMR), DEPT, MS and two-dimensional correlation (H-H COSY, Hetero-nuclear Single Quantum Correlation (HSQC), HMBC, NOE and NOESY) studies as 7ß,15α-dihydroxyl-lup-20(29)-ene-3ß-esters of palmitic (I), stearic (II) and eicosanoic acids (III). The characterisation of other isolated compounds is ongoing. Remarkably, this is the first report of the existence of fatty acid esters of an unusual 7ß,15α-dihydroxylated lupeol in the Eastern Nigeria mistletoe. These isolated compounds might contribute in part to the numerous established bio-activities of the Eastern Nigeria mistletoes.

A novel sesquiterpene acid and an alkaloid from leaves of the Eastern Nigeria mistletoe, Loranthus micranthus with potent immunostimulatory activity on C57BL6 mice splenocytes and CD69 molecule.

CONTEXT: The Eastern Nigeria mistletoe, Loranthus micranthus Linn. (Loranthaceae), is used in the treatment of several diseases including immune-modifying diseases and thus there is a need to identify the immunoactive constituents. OBJECTIVE: This research isolated and characterized the immunoactive constituents in the Eastern Nigeria mistletoe. MATERIALS AND METHODS: Bioassay-guided fractionation was employed in the isolation and purification of the constituents. The characterized compounds were screened for immunostimulatory activities on isolated C57BL/6 mice splenocytes and early activation marker, CD69 at concentrations of 10, 25, and 100 µg/mL using flow cytometry techniques and compared to lipopolysaccharide (LPS; 10 µg/mL) and concanavalin A (ConA; 2 µg/mL) as standards. RESULTS: Two compounds, a novel sesquiterpene, 2, 3-dimethoxy-benzo [a, b] cyclopentenyl-3',3',5'-trimethyl pyran-4-carboxylic acid, and a known alkaloid, lupinine were isolated and characterized. The compounds (25 µg/mL) showed statistically significantly (p < 0.05) stimulatory activity on the splenocytes with values of 56.34 ± 0.26% and 69.84 ± 0.19%, respectively, compared to 7.58 ± 0.42% recorded for the unstimulated control. Similarly, the CD69 expression assay showed immunostimulation with statistically significant values (p < 0.05) of 2.31 ± 0.07% and 2.71 ± 0.03%, respectively, compared to 1.69 ± 0.05% recorded for the nonstimulated control. DISCUSSION: These data suggest that the isolated compounds possess immunomodifying abilities. In addition, the activation of the CD69 molecule is possibly one of its mechanisms of action. CONCLUSION: These compounds may be responsible in part, for the immunostimulatory activities already established for the Eastern Nigeria mistletoes.

Immunomodulatory activity of a lupane triterpenoid ester isolated from the eastern Nigeria mistletoe, Loranthus micranthus (Linn).

OBJECTIVE: To provide further evidence for the ethnomedicinal use of the Eastern Nigeria mistletoe, Loranthus micranthus (L. micranthus), as an immunostimulant. METHODS: Solvent fractions from the crude extract of the mistletoe plant was obtained and screened by the cell mediated delayed type hypersensitivity reaction (DTHR) model in mice. Then the immunomodulatory potentials of a major lupane triterpenoid ester isolated from an active hexane fraction of the Eastern Nigeria mistletoe was investigated. Three lupeol-based triterpenoid esters: 7ß 15α-dihydroxyl-lup-20(29)-ene-3ß-palmitate (I), 7ß, 15α-dihydroxyl-lup-20(29)-ene-3ß-stearate (II) and 7ß, 15α-dihydroxyl-lup-20(29)-ene-3ß-decadecanoate (III) were isolated from the plant leaves epiphyting on a local kola nut tree and were characterized. Compound 1 was subjected to cell proliferation studies using C57Bl/6 splenocytes at three dose levels (5, 25 and 100 µg/mL) in presence of controls. Furthermore, the effect of this compound on IL-8 receptor expression was evaluated at three doses (1, 5 and 10 µg/mL) using the real time polymerase chain reaction assay. RESULTS: This triterpenoid ester produced some enhancement of the splenocytes at the tested doses but at doses higher than 5 µg/mL caused inhibition of the IL-8 receptor expression. CONCLUSIONS: The present findings support the ethnomedicinal use of the Eastern Nigeria Mistletoe in the management of diseases affecting the immune system. The triterpenoid(s) have some immunomodulatory abilities on splenocytes and IL-8 receptors and may partly account for the overall immunomodulatory activity of this plant.

Uncovering the therapeutic potential of natural products with biomarker-guided screening.

Biomarker-guided screening is a multidisciplinary approach that can be used to facilitate early drug discovery from natural products. By integrating powerful genomic tools and the rapidly expanding knowledge of biomarkers, the screening systematically identifies compounds with therapeutic potential from herbal medicines. Compounds identified through this screening are associated with biomarkers, which provide unique bases for the further characterization of the therapeutic potential, as well as biological mechanisms, of these molecules. This feature review outlines the basic concept of biomarker-guided screening, and describes two proof-of-principle studies, which demonstrated the promise and technical feasibility of this methodology. The scope and limitations of this method are also discussed.

A polyacetylene compound from herbal medicine regulates genes associated with thrombosis in endothelial cells.

From Toki-shakuyaku-san, an herbal formulation for 'cleansing stagnated blood,' a key gene regulatory compound was purified and identified through a screening based on DNA microarray and real-time PCR.

Rediscovery of natural products using genomic tools.

A screening methodology called 'genomic screening' was established to identify natural products that can regulate cellular gene expression. Application of genomic screening to Keishi-bukuryo-gan (KBG), a Japanese herbal medicine formulation, identified a previously unnoticed transcriptional effect by linoleic acid, a known KBG component. The approach opens up a possibility to develop cell-permeable molecular tools for functional genomics research and sets a stage to evaluate the potential of natural products for transcription therapies.

Nocturnal gastric acid breakthrough during the administration of rabeprazole and ranitidine in Helicobacter pylori-negative subjects: effects of different regimens.

BACKGROUND: Nocturnal gastric acid breakthrough (NAB) is defined as nocturnal intragastric pH less than 4 for more than 1 h during proton pump inhibitor (PPI) administration. A bedtime dose of an H2 receptor antagonist (H2RA) inhibites NAB, but the efficacy of the H2RA decreases with continuous administration. We carried out the present study to investigate the effect of 14-day H2RA administration on NAB. METHODS: Ten male volunteers without Helicobacter pylori infection received four different 14-day regimens of rabeprazole and ranitidine (study a, morning dose of 20 mg rabeprazole; study b, morning dose of 20 mg rabeprazole with a single bedtime dose of 150 mg ranitidine only on the last day; study c, continuous 20 mg morning dose of rabeprazole and 150 mg at bedtime; study d, morning and evening doses of 10 mg rabeprazole). Ambulatory 24-h gastric pH monitoring was conducted on the last day of each regimen. RESULTS: NAB in studies a, b, c, and d was observed in 9, 1, 4, and 4 subjects, respectively, and the longest periods of nocturnal gastric pH at less than 4.0 were 102.5, 14.0, 37.5, and 52.5 min, respectively (study b vs study c, P<0.05). CONCLUSIONS: The continuous inhibitory effect of ranitidine combined with rabeprazole on nocturnal gastric acid secretion declined during 14-day-long administration in H. pylori-negative subjects. Split dosing of rabeprazole was more effective than the single morning dose for inhibiting nocturnal gastric acid secretion.