Using commercial nutraceutical mixes as immune stimulants: an in vitro proliferation study using Metabolic Cell-Support[TM] on non-stimulated human lymphocytes.
Traditional medicine has long utilised the medicinal properties of mushrooms and herbs for healing and health. Scientific validation of the clinical efficacy of these natural therapies has resulted in a commercial approach to delivery, including the pharmaceutical preparation of active ingredients into both capsules and tablets. Unfortunately this approach has in some circumstances resulted in products that when compared to their natural sources have lost functionality (Borchers 2004).
This study examines a commercially available immune stimulate, Metabolic Cell-Support[TM] (MCS) to determine whether its herbal based formulation retains immunological activity comparable to what is observed for its individual natural ingredients.
Metabolic Cell-Support[TM] is composed of three medicinal mushrooms, Ganaderma Lucidum, Lentinus edodes (mycelia) and Coriolus versicolor; a herb, Astragalus membranaceus (from the root) and vitamin C. Individually all these components have demonstrated immune stimulating potential but using combinations of these has not been extensively tested.
It is known that a range of active compounds found in herbs can stimulate the human immune system. As these compounds can vary in bioactivity and biospecificity it may be possible to utilise a range of the compounds to symbiotically stimulate the immune system leading to a more efficacious response (Smith 2002). The formulation of MC-S has been based upon this principle.
Ganaderma lucidum (lingzhi or reishi) has long been regarded in traditional Chinese medicine as enhancing body disease resistance and consolidating the constitution of patients (Lin ZB 2005). Studies on the polysaccharide extracts of Ganaderma lucidum have demonstrated mitogenicity and activation of immune effector cells such as macrophages, natural killer cells and T-lymphocytes (Goa 2001), stimulating the production of cytokines interferon (INF), interleukins (IL) and tumor necrosis factor (TNF).
Lentinus edodes (shiitake mushroom) is a common edible mushroom. The two extracts: LEM extract (from Lentinus edodes mycelium) and Lentinan are both proven immunomodulators augmenting activation and proliferation of peripheral mononuclear cells (Aoki 1984, Hobbs 2000).
Coriolus versicolor contains two bioactive polysaccharides that are chemically similar, polysaccharide-peptide (PSP) and polysaccharide-K or krestin (PSK). These only differ by the presence of fucose on PSK and rhamnose and arabinose in PSP (Smith 2002). Both are potent immunomodulating activating T-cells, antigen presenting cells, monocytes and macrophages, and induce INF gamma and IL2 production (Tzianabos 2000). Finally, extracted polysaccharides and saponins from Astragalus membranaceus have been shown both in vitro and in vivo to stimulate NK-cell activity and PBL proliferation (Sun 1983).
The objective of this study was to examine the effect of varying doses of MC-S on the in vitro proliferation of non stimulated human peripheral blood lymphocytes (PBLs) as a measure of immune stimulating potential.
Materials and Methods
Metabolic Cell-Support (MC-S)
MC-S (without vitamin C) (Metabolic Research Pty Ltd, Newcastle, Australia) was used in powdered form. The vitamin C was excluded from the in vitro testing due to its acidity and potential inhibitory effect on the tissue culture. Prior to testing a stock solution of MC-S was prepared in sterile water (100,000 * mg [mL.sup.-1]).
The test material was then prepared by diluting this stock to a final concentration of 781.25 * mg [mL.sup.-1] in AIM-V serum free medium (Gibco, Invitrogen, Carlsbad California USA) and sterile filtering through a 0.22 micron filter (Sartorius, Hannover Germany). Stimulating efficacy of this test material was studied using 1:4 dilutions to a minimum concentration of 0.0004 [micro]mg [mL.sup.-1] in AIM-V serum free medium.
Peripheral blood lymphocyte (PBL) preparation
Human whole blood was defibrinated using universal tubes containing 4 mm glass beads and PBLs were collected by density gradient centrifugation separation. In brief, 20 mL of defribinated blood diluted 1:2 with Hanks buffered salt solution (HBSS) was overlayed on 15 mL of Ficoll-Paque[TM] Plus (Amersham Biosciences Uppsala, Sweden) and centrifuged (Sigma 3-16k Centrifuge, Sigma Laborzentrifugen Osterode, Germany) at 400 g for 40 minutes at 20[degrees]C. Following centrifugation the PBL rich interface was harvested and pelleted by centrifugation. The PBL harvest was then washed twice with Hanks buffered salt solution (HBSS) and resuspended in AIM-V serum free media at a final concentration of 3x[10.sup.6] cells [mL.sup.-1].
Lymphocyte proliferation assay
An amount of 50 [micro]mL of each dilution of MC-S test solution was added to 6 wells of a 96 well flat bottom culture plate (Nunc, Roskilde Denmark). To three wells of each dilution set, 50 [micro]mL of PBL cell suspension was added giving a final concentration of 1.5x[10.sup.5] cells per well. In the remaining three wells for each dilution set was added 50 [micro]mL of AIM-V serum free media as a background control.
Controls for this assay were non treated non stimulated PBLs (negative control and base line) and non treated phytohemagglutinin (PHA) (Sigma, Sigma-Aldrich St Louis USA) stimulated PBLs 10[micro]g [ml.sup.-1] (positive control). Plates were incubated at 37[degrees]C in a C[O.sup.2] incubator for 48 hours. Proliferation was measured using Cell Titer 96 Aqueous Non-Radioactive Cell proliferation assay kit (Promega Corporation, Madison Wisconsin USA) as per manufacturers' instructions with absorbance measured at 490 nm using a Bio-Rad microplate reader model 680 (Bio-Rad Laboratories Hercules California USA). Absorbance data was calculated as a percentage of proliferation in comparison to non treated non stimulated control. Results were graphed as mean [+ or -] SD of 3 separate assays.
Statistical analysis was performed using T-test analysis for two samples assuming equal variances, two tailed using means on percentage of increase of each assay (Microsoft Excel). A p-value of less than 0.05 was considered to be significant.
The exposure of non stimulated PBLs to varying doses of MC-S over a 48 hour period mediated a proliferation response at each dose. The peak of proliferation for these experiments occurred at 1.526 [micro]g [mL.sup.-1] (Fig 1). Significant different proliferations were reported for the dose range of 0.024 [micro]g [mL.sup.-1] to 390.63 [microg [mL.sup.-1] (Table 1).
[FIGURE 1 OMITTED]
These experiments demonstrated that MC-S has a mitogenic effect on non stimulated PBLs, exhibiting a statistically significant peak proliferation of 29.7 [+ or -] 7.99% at a dose of 1.526 [micro]g/mL when compared to resting PBLs (p [less than or equal to] 0.003).
These findings compare favourably with the results achieved for the individual medicinal components of MC-S shown in other studies, however as most research to date in this area has been directed specifically at concentrates of active compounds isolated from the whole fungi and plant it is not possible to make direct comparisons. In this study the concentration of the known active compounds would be relatively low, especially considering that 200 kg of shiitake mushrooms yields only 31 g of Lentinan (Smith 2000). This makes the results of the current study more significant.
Although the clinical efficacy of the individual components of MC-S has been long recognised and utilised in Asian countries, the Western world has only in recent years realised the potential of complementary medicines. Alongside this realisation is the acknowledgement of the need for scientific validation of potential clinical applications. In Australia the Therapeutic Goods Administration (TGA) does monitor claims made for complementary therapies, but a gap still exists between accepted applications of whole or parts of plants and fungi, isolated active agents and combinations of both. When these products are turned into pharmaceutical preparations, once again their efficacy requires re-testing. Unfortunately many products are in the market without what could be considered adequate scientific review.
This study does demonstrate that MC-S actively proliferates immune cells in vitro, suggesting that as a commercial product it stimulates immune activity. Future clinical studies are likely to support this claim.
This study was undertaken at the University of Newcastle, Callaghan Australia. The authors would like to acknowledge however that the funding for this study was provided by Metabolic Health Pty Ltd (Newcastle Australia).
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Corresponding author DA Clark email: Daveaclark@hotmail.com
TUNRA Limited, Industry development Centre, University Drive, Callaghan NSW 2308 Australia
School of Environmental and Life Sciences, Faculty of Science and IT, University of Newcastle,
Callaghan NSW 2308 Australia
Table 1 Comparative analysis of PBL proliferation at each dose of MC-STM between treated samples and non-treated control. Results are representative of 3 experiments performed in triplicate. Percentage of Dose [micro]g/mL proliferation [+ or -] SD p value 0.0004 4.5 [+ or -] 6.14 0.273 0.0015 8.23 [+ or -] 6.4 0.088 0.006 16.5 [+ or -] 10.44 0.051 0.024 22.57 [+ or -] 6.21 0.003 0.095 23.97 [+ or -] 4.8 0.0009 0.381 26.47 [+ or -] 3.17 0.0001 1.526 29.67 [+ or -] 8 0.003 6.104 14.93 [+ or -] 9 0.049 24.414 20.83 [+ or -] 3.31 0.0004 97.656 13.83 [+ or -] 6.64 0.022 390.63 16.83 [+ or -] 7.12 0.015
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|Author:||Clark, D.A.; Adams, M.C.|
|Publication:||Australian Journal of Medical Herbalism|
|Date:||Sep 22, 2007|
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