Archives: Projects

Portfolio Items

Immune modulating activity of a mycelial-based medicinal mushroom is a combination of the mycelium itself and compounds in the fermented substrate.

Benson KF,1 Stamets P,2 Davis R,2 Taylor A,2 Jensen GS.1
1 NIS Labs, Klamath Falls, Oregon, USA.
2 Fungi Perfecti, Olympia, Washington, USA.

Many medicinal mushroom products are manufactured for consumption as a crude powder from mycelium and its fermented substrate. While pre-clinical and clinical studies have been performed on these products, the immunological contributions of the fermented substrates have not been examined. The goal for this study was to evaluate the immune-modulating properties of the mycelium versus the fermented substrate, to document whether an important part of the immune-activating effects resides in the metabolically converted, fermented substrate.

The medicinal mushroom Trametes versicolor (Tv, Turkey Tail) is one of the fungi that is often prepared as a powder from the complex mass of the fungal mycelium and the fermented substrate on which it grew. Tv was selected as a model organism for this testing, because the physical structure of the mycelium is well-defined, and can be mechanically separated from its substrate, which allows for harvest of both the mycelium and the fermented substrate when cultivated on a loose substrate such as rice flour.

Tv mycelium was inoculated and cultured on finely milled rice flour. Subsequently, the mycelium was mechanically separated from the substrate, and both the mycelium and the fermented substrate were dried and milled to a powder. A sample of the initial rice flour substrate served as a control. Each of the three powders were used to generate aqueous extracts without heating, which were filtered through 0.22-micron filters to remove particulate matter. The solid fractions were then passed through homogenization spin columns, but not filtered.
Peripheral venous blood samples were obtained from healthy volunteers after providing written informed consent, approved by a registered IRB board (FWA 2603). The aqueous and solid fractions of the initial substrate, the fermented substrate, and the Tv mycelium were tested for immune-activating and modulating activities on human peripheral blood mononuclear cell cultures, to examine induction of the expression of the CD69 activation marker on lymphocytes versus monocytes, and on the T, NKT, and NK lymphocyte subsets. Culture supernatants were tested for cytokines using Luminex magnetic bead arrays and the MagPix® multiplexing system.

Both the aqueous and solid fractions of the mycelium triggered robust induction of CD69 on lymphocytes and monocytes, whereas the fermented substrate only triggered minor induction of CD69. The aqueous extract of the mycelium had a stronger activating effect than the solid fraction. In contrast, the aqueous extract of the initial substrate had no effect on CD69 expression, whereas the solid fraction of the initial substrate triggered a reduction in CD69, below that of untreated cells.
Both the aqueous and solid fractions of the fermented substrate triggered large and dose-dependent increases in immune-activating pro-inflammatory cytokines (IL-2, IL-6), the anti-inflammatory cytokines Interleukin-1 receptor antagonist (IL-1ra) and Interleukin-10 (IL-10), the anti-viral cytokines interferon-gamma (IFN-γ) and Macrophage Inflammatory Protein-alpha (MIP-1α), as well as Granulocyte-Colony Stimulating Factor (G-CSF) and Interleukin-8 (IL-8).
The mycelium triggered modest effects on cytokines, and with stronger effects at lower doses. For all eight cytokines, the effects of the lowest dose of the aqueous extract of the mycelium exceeding that of the same dose of the aqueous extract of the fermented substrate.
In contrast, the aqueous extract of the initial substrate showed no effects on cytokine induction, whereas the solid fraction showed some modest effect on induction of cytokines and growth factors.

The results have demonstrated that the immune-activating bioactivity of mycelial-based medicinal mushrooms are a combination of the mycelium itself (including insoluble beta-glucans, but also water-soluble components), and the fermented substrate containing highly bioactive metabolites that are absent in the initial substrate.

KFB and GSJ are employed at NIS Labs, an independent contract research organization specializing in natural products research, and have no financial interests in the subject matter. RD and AT are employed by the sponsor of the study. PS holds several patent applications on topics related to the presented work, and is the founder and owner of the sponsoring company. The study was sponsored by Fungi Perfecti, LLC, a grower and producer of commercially available mushroom mycelia and finished consumable products.

Torkelson CJ, Sweet E, Martzen MR, Sasagawa M, Wenner CA, Gay J, Putiri A, Standish LJ: Phase 1 Clinical Trial of Trametes versicolor in Women with Breast Cancer. ISRN Oncol 2012, 2012:251632.

Inactivated probiotic Bacillus coagulans GBI-30 induces complex immune activating, anti-inflammatory, and regenerative markers in vitro

Objective: The aim of this study was to document the immune activating and anti-inflammatory effects of inactivated probiotic Bacillus coagulans GBI-30, 6086 (Staimune™) cells on human immune cells in vitro.
Methods: In vitro cultures of human peripheral blood mononuclear cells (PBMC) from healthy blood donors were treated with inactivated B. coagulans GBI-30, 6086 cells for 24 hours. After incubation, the PBMC were stained with fluorochrome-labeled monoclonal antibodies for CD3, CD56, and CD69 to monitor cellular activation by flow cytometry. The culture supernatants were tested for cytokine profile using a 27-plex Luminex array, including pro- and anti-inflammatory cytokines, chemokines, and growth factors.
Results: Inactivated B. coagulans GBI-30, 6086 cells induced the CD69 early activation marker on CD3+ CD56− T lymphocytes, CD3+ CD56+ NKT cells, CD3−CD56+ NK cells, and also some cells within the CD3−CD56− non-T non-NK cell subset. Culture supernatants showed robust increases in the immune-activating cytokines IL-1β, IL-6, IL-17A, and TNF-α. IFN-γ levels were increased, along with three chemokines, MCP-1, MIP-1α, and MIP-1β. The two anti-inflammatory cytokines IL-1ra and IL-10 showed increases, as well as the G-CSF growth factor involved in repair and stem cell biology. In contrast, GM-CSF levels showed a mild decrease, showing a highly selective growth factor response.
Conclusion: The inactivated B. coagulans GBI-30, 6086 cells activated human immune cells and altered the production of both immune activating and anti-inflammatory cytokines and chemokines. Of special importance is the novel demonstration of a selective upregulation of the G-CSF growth factor involved in postinjury and postinflammation repair and regeneration. This suggests that important immunogenic cell wall components, such as lipoteichoic acid, are undamaged after the inactivation and retain the complex beneficial biological activities previously demonstrated for the cell walls from live B. coagulans GBI-30, 6086 (GanedenBC30) probiotic bacteria.

Read More

Antioxidant and Immune-Modulatory Effects of Aloe vera Decolorized Leaf Juice vs. Inner Leaf Juice

The aloe vera (Aloe vera) plant has a long history of traditional therapeutic topical use. Its leaf and inner leaf juices are also ingested in various forms. Aloe vera leaf juice is made from macerated aloe leaves, and when it is intended for human ingestion, it is purified of latex constituents via a charcoal filtration process known as decolorization. Several publications have shown immunomodulatory effects, as well as antibacterial, antifungal and anti-parasitic properties of aloe vera inner leaf juice, but limited information is available regarding these effects for aloe vera leaf juice after the decolorization process. The aim of this preliminary study was to compare biologically-relevant antioxidant and immune-modulatory effects of aloe vera inner leaf and decolorized leaf preparations using a variety of in vitro techniques.

ExpBio poster 5-1-17


Water-soluble egg membrane enhances the immunoactivating properties of an Aloe vera-based extract of Nerium oleander leaves

Objective: To evaluate a blend of two natural ingredients on immune parameters relevant for their current topical use and potential support of microcirculation in skin tissue.

Materials and methods: A blend (BL) of Aloe vera-based Nerium oleander extract (NAE-8i, oleandrin-free) and hydrolyzed water-soluble egg membrane (WSEM) was applied to human whole-blood cultures for 24 hours, with each separate ingredient serving as a control. Immune-cell subsets were analyzed for expression levels of the activation markers CD69 and CD25. Culture supernatants were analyzed for cytokines, chemokines, and immunoregulating peptides.
BL increased CD69 expression on lymphocytes, monocytes, and CD3–CD56+ natural killer cells, and CD25 expression on natural killer cells. The number of CD69+CD25+ lymphocytes increased in cultures treated with BL and the separate ingredients. BL triggered production of multiple cytokines and chemokines, where CC chemokines MIP1α and MIP3α, as well as cytokines involved in wound healing – Groα, Groβ, ENA78, and fractalkine – reached levels manyfold above treatment with either NAE-8i or WSEM alone.
Conclusion: Data on BL showed that WSEM strongly enhanced NAE-8i’s effects on immunoactivation in vitro. This has potential relevance for support of immunity in skin tissue, including antibacterial and antiviral defense mechanisms, wrinkle reduction, and wound care.

Read more

Reduction of facial wrinkles by hydrolyzed water-soluble egg membrane associated with reduction of free radical stress and support of matrix production by dermal fibroblasts

Objective: The aim of this study was to evaluate the effects of water-soluble egg membrane (WSEM) on wrinkle reduction in a clinical pilot study and to elucidate specific mechanisms of action using primary human immune and dermal cell-based bioassays.
Methods: To evaluate the effects of topical application of WSEM (8%) on human skin, an open-label 8-week study was performed involving 20 healthy females between the age of 45 years and 65 years. High-resolution photography and digital analysis were used to evaluate the wrinkle depth in the facial skin areas beside the eye (crow’s feet). WSEM was tested for total antioxidant capacity and effects on the formation of reactive oxygen species by human polymorphonuclear cells. Human keratinocytes (HaCaT cells) were used for quantitative polymerase chain reaction analysis of the antioxidant response element genes Nqo1, Gclm, Gclc, and Hmox1. Evaluation of effects on human primary dermal fibroblasts in vitro included cellular viability and production of the matrix components collagen and elastin.
Results: Topical use of a WSEM-containing facial cream for 8 weeks resulted in a significant reduction of wrinkle depth (P<0.05). WSEM contained antioxidants and reduced the formation of reactive oxygen species by inflammatory cells in vitro. Despite lack of a quantifiable effect on Nrf2, WSEM induced the gene expression of downstream Nqo1, Gclm, Gclc, and Hmox1 in human keratinocytes. Human dermal fibroblasts treated with WSEM produced more collagen and elastin than untreated cells or cells treated with dbcAMP control. The increase in collagen production was statistically significant (P<0.05).
Conclusion: The topical use of WSEM on facial skin significantly reduced the wrinkle depth. The underlying mechanisms of this effect may be related to protection from free radical damage at the cellular level and induction of several antioxidant response elements, combined with stimulation of human dermal fibroblasts to secrete high levels of matrix components.

Read More

Consumption of nattokinase is associated with reduced blood pressure and von Willebrand factor, a cardiovascular risk marker: results from a randomized, double-blind, placebo-controlled, multicenter North American clinical trial

Objective: The objective of this study is to evaluate the effects of consumption of nattokinase on hypertension in a North American hypertensive population with associated genetic, dietary, and lifestyle factors. This is in extension of, and contrast to, previous studies on Asian populations.
Materials and methods: A randomized, double-blind, placebo-controlled, parallel-arm clinical study was performed to evaluate nattokinase (NSK-SD), a fermented soy extract nattō from which vitamin K2 has been removed. Based on the results from previous studies on Asian populations, 79 subjects were enrolled upon screening for elevated blood pressure (BP; systolic BP ≥130 or diastolic BP ≥90 mmHg) who consumed placebo or 100 mg nattokinase/d for the 8-week study duration. Blood collections were performed at baseline and 8 weeks for testing plasma renin activity, von Willebrand factor (vWF), and platelet factor-4. Seventy-four people completed the study with good compliance.
Results: Consumption of nattokinase was associated with a reduction in both systolic and diastolic BP. The reduction in systolic BP was seen for both sexes but was more robust in males consuming nattokinase. The average reduction in diastolic BP in the nattokinase group from 87 mmHg to 84 mmHg was statistically significant when compared to that in the group consuming placebo, where the average diastolic BP remained constant at 87 mmHg (P<0.05), and reached a high level of significance for males consuming nattokinase, where the average diastolic BP dropped from 86 mmHg to 81 mmHg (P<0.006). A decrease in vWF was seen in the female population consuming nattokinase (P<0.1). In the subpopulation with low plasma renin activity levels at baseline (<0.29 ng/mL/h), an increase was seen for 66% of the people after 8-week consumption of nattokinase (P<0.1), in contrast to only 8% in the placebo group.
Conclusion: The data suggest that nattokinase consumption in a North American population is associated with beneficial changes to BP in a hypertensive population, indicating sex-specific mechanisms of action of nattokinase’s effect on vWF and hypertension.

Read more

Consumption of an aqueous cyanophyta extract derived from Arthrospira platensis is associated with reduction of chronic pain: results from two human clinical pilot studies

Objectives: The aim of this study was to evaluate the effects of consumption of an aqueous cyanophyta extract (ACE) from Arthrospira platensis on chronic pain in humans, in two clinical pilot studies.

Design and interventions: The two pilot studies each involved 12 subjects experiencing chronic pain. The initial study followed an open-label 4-week study design involving consumption of 1 g ACE per day. A subsequent placebo-controlled, single-blind, crossover study involved consumption of 500 mg ACE, 250 mg ACE, or 0 mg ACE (placebo) per day for 1-week duration, separated by 1-week washout period.
Subjects: Adult subjects of both sexes, with chronic joint-related pain for at least 6 months prior to enrollment, were recruited after obtaining written informed consent.
Outcome measures: Visual analog scales were used to score pain at rest and during physical activity for each person’s primary and secondary areas of chronic pain. An activities of daily living questionnaire was used to collect data on physical functioning.
Results: The data showed rapid reduction of chronic pain in people consuming ACE, where the reduction in pain scores for each person’s primary pain area reached a high level of statistical significance after 2 weeks of consumption (P<0.01), both when at rest and when being physically active. Secondary pain areas when physically active showed highly significant improvements within 1 week of consumption of 1 g/d (P<0.001) and borderline significant improvements within 1 week of consuming 500 mg/d (P<0.065) and 250 mg/d (P<0.05). This was accompanied by an increased ability to perform daily activities (P<0.05). A small but significant weight loss was observed during the 4-week study, as the average body mass index dropped from 31.4 to 29.4 (P<0.01).
Conclusion: Consumption of ACE was associated with reduction of chronic pain, as well as a dose-dependent increased ability to perform activities of daily living.

Read more

Reduction of body fat and improved lipid profile associated with daily consumption of a Puer tea extract in a hyperlipidemic population: a randomized placebo-controlled trial

Objective: The goal for this study was to evaluate the effects of daily consumption of Puer tea extract (PTE) on body weight, body-fat composition, and lipid profile in a non-Asian population in the absence of dietary restrictions.
Materials and methods: A randomized, double-blind, placebo-controlled study design was used. A total of 59 overweight or mildly obese subjects were enrolled upon screening to confirm fasting cholesterol level at or above 220 mg/dL (5.7 mmol/dL). After giving informed consent, subjects were randomized to consume PTE (3 g/day) or placebo for 20 weeks. At baseline and at 4-week intervals, blood lipids, C-reactive protein, and fasting blood glucose were evaluated. A dual-energy X-ray absorptiometry scan was performed at baseline and at study exit to evaluate changes to body composition. Appetite and physical and mental energy were scored at each visit using visual analog scales (0–100).
Results: Consumption of PTE was associated with statistically significant weight loss when compared to placebo (P<0.05). Fat loss was seen for arms, legs, and the gynoid region (hip/belly), as well as for total fat mass. The fat reduction reached significance on within-group analysis, but did not reach between-group significance. Consumption of PTE was associated with improvements to lipid profile, including a mild reduction in cholesterol and the cholesterol:high-density lipoprotein ratio after only 4 weeks, as well as a reduction in triglycerides and very small-density lipoproteins, where average blood levels reached normal range at 8 weeks and remained within normal range for the duration of the study (P<0.08). No significant changes between the PTE group and the placebo group were seen for fasting glucose or C-reactive protein. A transient reduction in appetite was seen in the PTE group when compared to placebo (P<0.1).
Conclusion: The results from this clinical study showed that the daily consumption of PTE was associated with significant weight loss, reduced body mass index, and an improved lipid profile.

Read more

The Effect of Consumption of a Nopal Cactus Fruit Juice on C-Reactive Protein Levels in Healthy Adults: Results from a Randomized, Double-Blind, Controlled Clinical Pilot Study

The Nopal cactus grows widespread in many parts of the world, and in some areas is considered a noxious weed. In addition to the use of the stems and leaves (also called pads or cladodes) and fruits (prickly pears) as food, Nopal has a long use in traditional folk medicine. The fruit shares some constituents with the stems, and contains additional unique polyphenols and betalain pigments with antioxidant and anti-inflammatory activities. A randomized, double-blind, controlled, parallel-arm human study was performed to compare consumption of Nopal fruit juice (NFJ) to control (apricot) juice. Serum C-reactive protein (CRP) and uric acid were measured at baseline and after 8 and 12 weeks. Reduced CRP levels were seen in both the NFJ and control groups. The 21.0% reduction at 8 weeks in the NFJ group was significantly more robust than the 6.6% reduction in the control group (P <.05). The reduced CRP level within the NFJ group was highly significant at 8 weeks (P <.0001), and remained reduced by 10.6% at 12 weeks compared to baseline (P <.065). The CRP reduction was not significant within the control group. There was no significant difference in uric acid levels between the groups at either 8 or 12 weeks. The 5.2% increase in uric acid levels within the control group was highly significant at 8 weeks (P <.0003), remaining significant at 12 weeks (P <.04). In contrast, a transient 3.6% increase in uric acid in the NFJ group at 8 weeks (P <.02) returned almost back to baseline levels at 12 weeks. Consumption of NFJ was associated with significant reduction in the CRP inflammatory biomarker while maintaining uric acid well within healthy range.

Read More

Aloe macroclada from Madagascar Triggers Transient Bone Marrow Stem Cell Mobilization

Objective: Aloe has been used for the treatments of various ailments dating back almost 6000 years. There are more than 450 species of aloe coming from various parts of Africa and South America, and from the island of Madagascar that contains unique species endemic to the island. One such species is Aloe macroclada that has been used for centuries by the local residents as a remedy for a wide variety of ailments. We investigated whether the mechanism of action behind the wide-ranging health benefits of A. macroclada could be mobilization of bone marrow stem cells.
Methods: A. macroclada was prepared into small spherical pellets by Malagasy healers using traditional methods of fabrication. The traditional dose of three pellets was fed to 4 volunteers and the number of circulating stem cells was quantified 1, 2 and 3 hours after consumption using flow-cytometry.
Results: The usual dose and preparation of A. macroclada traditionally used in Madagascar triggered a significant increase (up to 53%) in the number of circulating CD45dim CD34+ and CD34+ CD133+ stem cells within 2 hours of consumption. This increase lasted more than 3 hours and was significant after 120 and 180 minutes of consumption.
Conclusion: Consumption of A. macroclada has been credited with significant improvements in a wide variety of heath conditions. This data suggest that stem cell mobilization may be an important mechanism of action behind the health benefits of A. macroclada.

Read More