2. Essential Fungiflex methods
Most of the methods and materials that we used in this research are detailed in the printed versions of this report; but there are two aspects that we need to stress right at the start: the identity of the organism we used and the basic method for extracting the bioactive materials we called Fungiflex.
Organism
Most of the work was done with the ink cap mushroom Coprinopsis cinerea. The organism has previously been called (by us and by our colleagues) Coprinus cinereus and before that Coprinus lagopus and Coprinus macrorhizus. It is widespread in temperate climates around the world being a commonly-encountered member of the community that recycles leaf litter. Its life cycle, genetics and methods for in vitro cultivation have been described by Moore & Pukkila (1985) [download a PDF of this publication into a new window by clicking here].
Extraction methods
Numerous methods for extraction of bioactive materials, involving a variety of organic solvents, hot and cold, and a wide range of salt solutions, have been described in the literature. So we want to stress at the outset that our method was the simplest of all; it simply consisted of steeping (or infusing) freshly harvested mushroom tissue in sterile distilled water at room temperature.
No vortexing, no mixing, no blending, no grinding.
After a few hours standing on the bench, the liquid was drained off the tissue, filtered and freeze-dried. The freeze-dried material was our bioactive extract.
Usually, the tissue we used was some part of a Coprinopsis mushroom fruit body; the nature and quantity of that tissue depended on the nature and purposes of the planned experiments, but in all cases the required mushrooms were freshly detached from the parent mycelium immediately prior to being placed in the steeping/infusing water. At various times and for different experiments the detached tissues may have been:
- at the stage of a young fruit body primordium, which was 1 to 3 mm in diameter and height, and harvested before the time of meiosis (= pre-meiosis);
- an older, differentiated fruit body primordium (more than 3 mm in diameter and height), immature, and though cap and stipe tissues were clearly developed, these were also pre-meiosis;
- or a young fruit body, which was 20 to 50 mm in height, the cap of which was still cylindrical (that is, still enclosing the apex of the stipe, and not expanded like a coprinoid ‘umbrella’); the basidia of this caps had completed the meiotic division (= post-meiosis), though the youngest in this class may not have yet formed spores, or spores, if formed, may not have developed their characteristic ‘black’ pigmentation (progress of meiosis, and spore formation and pigmentation were easily followed by light microscopy of squashes of tiny slivers of gill tissue).
Different extracts could be made by removing the caps from young fruit bodies, which could be done without damaging the stipe; in these experiments the caps and stipes were placed separately into pre-weighed and pre-sterilised 25 ml plastic tubes or pre-weighed, autoclaved 25 ml glass tubes.
Caps of primordia were left intact as removing them caused severe injury to both the cap and stipe. There were no differentiated zones in the youngest primordia and these were placed whole into pre-weighed sterilised plastic or glass tubes.
For each tissue sample used, the wet weight was determined and recorded. Sterilised distilled water was added to fill the tube completely (between 10 - 30 ml depending on the volume of tissue in the tube). Tubes were sealed tightly and kept in the dark at room temperature (21 - 23° C) for 1, 2 or 3 h.
As we were interested in gravitropism at the time, in some experiments comparisons were made between tubes that were kept vertical and those laid horizontal.
At the end of the infusion period the tissue(s) were removed from the tubes, the supernatant was filtered through Whatman No. 1 filter paper and then through a 0.45 μm cellulose nitrate membrane filter (Whatman) before being frozen at -20° C until needed.
Initially, when a number of tubes had been accumulated (volume more than 150 ml), they were frozen in liquid nitrogen individually and then set to freeze dry for 2-3 days. Later we found we could greatly reduce the volume of the pooled primary extract by rotary evaporation (water bath at less than 40° C) prior to being frozen in liquid nitrogen and freeze dried overnight.
Control experiments performed by infusing empty tubes with distilled water showed that there were no chemicals originating from the tubes or the filtration materials themselves which caused activities similar to those of our extracts.
Standard procedure
A standard procedure for analysis of a sample and development of the purification process is outlined as follows:
- Samples are bioassayed for Fungiflex 1 and Fungiflex 2 to verify their activity.
- Samples are fractionated either by reverse phase HPLC, silica gel chromatography or Thin Layer Chromatography (TLC).
- All fractions collected by chromatography are bioassayed to identify fractions in which activity is localised.
- Active fractions are then fractionated again by HPLC and/or TLC.
- Secondary fractions are bioassayed again and the process repeated until the compounds of interest are purified.
Outline of routine extraction and purification of Fungiflex growth hormones
Step 1
Water extraction of caps or stipes
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Methanol extraction (extract contains about 60 compounds)
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Reversed phase chromatography with a Partisphere SAX HPLC C 18 strong anion exchange column (about 30 compounds detectable at 254 nm)
Water: methanol (solvent gradient from 100% water to 100% methanol)
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Fraction 1 (3 min) = substances with growth inhibition and stimulation
activity (hydrophilic group)
Fraction 22+ (over 20 min) = substances with growth stimulation activity
(this is the hydrophobic group)
Step 2
C 18 Fraction 1
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Amino column chromatography (about 30 compounds)
80% Methanol: 20%water isocratic solvent system
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Peak 1 (3 min) = peak detectable at 254 nm, not containing bioactive substances
Peak 2 (4 min) = hexose positive peak detectable by colorimetric assay, containing bioactive substances
Step 3
C 18 Fraction 1
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Amino column chromatography (about 8 compounds)
Acetonitrile: water (65%: 35% to 60%: 40%) solvent gradient
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Peak 2 (4 min) = D-glucose
Peak 3 (6 min) = up to 3 substances with growth stimulation activity
Copyright © David Moore & Lily Novak-Frazer 2016
