Ganoderma lucidium tea

One Sunday I joined some mushroomers on a foray in the mountains.  We found some “reishi” mushrooms that have been used in Asian medicines for thousands of years.  Being something of a chemist, I wanted to know what was in the tea.
Wu DT, Deng Y, Chen LX, Zhao J, Bzhelyansky A, Li SP Evaluation on quality consistency of Ganoderma lucidum dietary supplements collected in the United States (2017)   Sci Rep. 7(1):7792.

This paper is available free on PMC

According to the authors, triterpenes and polysaccharides are generally considered to be the active ingredients in G lucidium fruiting bodies.

 

Like many of our steroid hormones, ganoderic acid is a cholesterol derivative.

The authors collected 19 different samples from places like Ebay and Amazon.  The products I found on Ebay do not come with a certificate of analysis. The biological activities of compounds with steroid hormone like structures may be different  from polysaccharides.

Triterpene extraction and analysis

Powder of each sample (1.0 g) was immersed in 20.0 mL of ethanol   and refluxed  for 30 min at 78 °C.  Ever Clear, 190 proof, is 95% ethanol.   Insoluble material was removed by centrifugation.  This is starting to sound like something an avid herbalist could do in his or her home lab.   Analysis differed from the home lab set up when the authors evaporated the extract to dryness and dissolved in 2 mL of methanol.

The authors used a high performance automated version (HPTLC) of thin layer chromatography.  TLC basically separates compounds by how much they adhere to an adsorbent material (stationary phase) as the solvent (mobile phase) flows up the material.

The plate was developed to a distance of 90 mm with dichloromethane/ethyl acetate/petroleum ether/formic acid/ethanol, 8:3:9:0.8:0.5 (v/v/v/v/v) as mobile phase at room temperature. Finally, the developed plates were colorized with 10% (v/v) H2SO4 in ethanol, and heated at 110 °C for 10 min.  The plates were photographed under white light and 365 nm UV light.

Sample GL20 is from an authenticated fruiting body.  The rather large range of bands is somewhat of a concern for the consumer wanting an authentic product.  An interesting follow up would be to grow the authenticated mushroom on different substrates, temperatures, humidity levels, oxygen tensions, and so on.

 

Polysaccharide extraction and analysis

Ethanol extraction sediments were extracted with pure water.

Potassium iodine (I2-KI) was used to colorize the polysaccharide extracts. It is not clear if the ethanol insoluble material was extracted once or twice or if the ethanol soluble material was analyzed for starch.  The first four columns are standards:

BK, water used as blank control

DN, malto-dextrin

ST, starch.

GL20 the reference control

Potassium iodine turns starch, glucose polymers blue.  Starch is a glucose polymer with 1-4 a-glycosidic bonds.   a-Amylase is used by brewers to digest starch to maltose, a glucose dimer.  According to the authors, the major bioactive polysaccharide in G. lucidum is  branched 1,3-β-D-glucan.  Like starch, these polysaccharides are glucose polymers.  Unlike starch they have  1-3 β-glycosidic bonds .

A polycaccharide fingerprint

Just a quick review of glycosidic bonds

Cellulose is a glucose polymer with beta-1,4 glycosidic bonds.  Starch is made up of amylose (alpha-1,4) and amylopectin ( a combination of alpha-1,4 and alpha-1,6) glycosidic bonds.
Polysaccharide mapping based on polysaccharide analysis using carbohydrate gel electrophoresis (PACE). The polysaccharide solution (1.0 mL) of each sample was mixed with α-amylase and 1,3-β-D-glucanase  and digested overnight at 40 °C. The hydrolysates
were dried and derivatized with 8-aminonaphthalene-1,3,6-trisulphonic acid (ANTS) for PACE analysis.

The authors performed hierarchical clustering to not only compare the number of  1,3-β-D-glucanase digest bands but also their relative intensity.

The closer the branches of the tree, the closer the samples are to one another.  GL09 and GL20, the reference, are very similar.

Other sugars

The discussion so far has focused on polymers of glucose.  Are there other sugars in the extracts?  The extracts were hydrolyzed with 2.0 M trifluoroacetic acid (1.0 mL) at 95 °C in a sealed tube for 10 h. The hydrolysates were washed with methanol and evaporated to dryness before derivation with hydroxylamine hydrochloride and acetic anhydride at 90 °C for 30 min.  Gas chromatography was used to resolve individual sugars.

These are examples of the 20 gas chromatograms in the publication.

These are Haworth projections of the four sugars with the pyranoses in the alpha configuration.

Determination of sizes of undigested polysaccharides

High performance size exclusion chromatography (HPSEC) was used to characterize the extracts.   To be more specific, the authors used “A rapid and accurate method for the quantitative estimation of natural polysaccharides and their fractions using high performance size exclusion chromatography coupled with multi-angle laser light scattering and refractive index detector. ”  To exclude the interference from the presence of additives such as starch or malto-dextrin, polysaccharides were treated with α-amylase before HPSEC.

 

HPSEC chromatograms of polysaccharides in G. lucidum dietary supplements before and after the  α-amylase digestion.

E Enzyme  α-amylase alone

GL Ganodermia lucidium supplement alone

GL + E, α-amylase digestion of the supplement

Note the large (blue) peak in GL18 before  α-amylase digestion.  GL09, which in other ways is similar to the reference, does not have the same high molecular weight glycan profile.

Authors’ conclusions

• Considered the major active ingredients in G lucidium extracts, Triterpenes and polysaccharides contents vary considerably in sources available to US consumers.
  derived from G. lucidum.
• Products should, at least have chemical characters of either triterpenes or polysaccharides based on their ethanol or water extract.
• The measured ingredients, only in 5 out of 19 (26.3%) tested samples, were in accordance with their labels.
• The results were similar to the data of DNA barcoding test reported in the Ne York Times in 2015.
• The quality consistency of G. lucidum dietary supplements collected in USA was extremely poor, which should be carefully investigated.
• The authors suggested that PACE carbohydrate mapping and size exclusion chromatography of undigested glycans may be an effective method of quality control.

From the perspective of a mushroom grower or harvester…

All of these methods seem to be very promising.  If one mushroom constitutes a year supply of reishi tea, the user might want to compare the glycan and triterpene profiles from the mushroom harvested one year with a second mushroom the next year.  Mushroom growers might be striving for a particular triterpene profile.  How do growth conditions affect this profile?

 

 

 

 

 

a very interesting brew