New Ergot Alkaloid Source?

[Bubbleplate]

While doing some research, I have run across what is potentially a breakthrough for a new, easy to produce source of Ergot Alkloids such as Ergotamine.
US Patent 2640007 (sorry no link) is entitled "Production of Alkaloids by Clitocybe Subilludens" and describes the culturing of the mushroom fungus Clitocybe subilludens, now known as Omphalotus olearius (Jack-O-Lantern mushroom)to produce Ergotamine, Ergonovine, etc. in large scale commercial production!
Omphalotus olearius is a common mushroom which grows on the roots of Oak and other hardwood trees, and also is  bioluminescent, i.e. it glows in the dark. Apparently this particular strain was originally found near Gainesville Florida growing on or near Canary Island palm trees. It is unique in that it is the only other source of ergot alkaloids other than in fungi that infect grass type plants like Claviceps.
Also interesting is that the patent authors discovered that the yield of mycelium may be increased substantially by utilizing fruit juice concentrates (i.e. Orange juice) in the culture medium. They also mention that the "type and quantity of the alkolids" may be influenced by "those skilled in the art that the well-known methods of producing mutations, such as the use of ultra-violet light or certain chemicals".
Omphalotus olearius is wide spread in North America. I would venture that by taking samples of many mushrooms, and testing with Ehrlich-van Urk reagent and paper chromotography/UV light, one could find a strain that produces Ergot alkaloids. (Particularly strains from Northern Florida!)
Omphalotus olearius is a very robust and hardy fungi, and submerged culture in large fermenters should be quite easy. It is nowhwere's near as fussy as Claviceps with regards to growing conditions.

http://www.ces.ncsu.edu/depts/hort/consumer/poison/Omphaol.htm

[pHarmacist]

Patent US2640007

You link to patent like this:

[patent]USpatent#[/patent]

[sean1234]

this is very interesting! i wonder what percentages of alkaloid contents are.

[abc_n_123]

Wow!
Very Interesting (maybe excellent) Find Bubbleplate!

I don't know about the rest of the U.S., but the Jack-O-Lantern is easily found in Southeast U.S. during the summer/fall (July through November, and I have read that it grows from November through March in California).  I used to show my friends the "glowing-shroom" during camping trips.  I almost always see a patch of Jack-O-Lanterns growing when I am hikeing in the fall.  Some unfortunate person might misidentify it for the Big Laughing Gym (Gymnopilus spectabilis, a psychedelic mushroom of the Eastern U.S.), but no worries if he does eat the Jack even though it is poisonous, because it would just end up in a couple days of stomach upset (never has happened to me   ).......enough with the chitchat.

I dug into this a little bit and I believe the mushroom we are looking for is really Omphalotus illudens and not O. olearius.  It is still a jack-O-lantern,it is just specific to the eastern U.S.  See the quote below. It is too bad that there were no expected yields or alkaloid contents in the patent. I am amazed that this mushroom may have a significant use.  It seems like I have seen it everywhere. It is very abundant!  Here is the interesting quote about O. olearius:

"Comments: Also called the False Chanterelle,and often mistaken for the Chanterelle, this is a species complex.  Some experts believe that the American forms may be distinct from the European form bearing the same name.  If so, the eastern Jack is more properly called O. illudens, and the western form, O. olivascens.  The eastern form has also been known as Clitocybe (sub-)illudens.  All forms are poisonous, typically causing gastric upset for a few hours to 2 days.  When this species is gathered fresh and taken into a dark room, the gills give off an eerie green glow."---National Audubon Society Field Guide to North American Mushrooms pg. 787

We will know whether or not this find is excellent or not when we can find the alkaloid content for O. illudens' mycelium specifically (Stating the obvious?   ). There probably is a write-up for O. olearius around somewhere, and even it says that there are no ergot alkaloids present it could be a different story for O. illudens if it is truly a separate species.  Come next fall, we will see what this mushroom has for us to play with.


These may have some interesting/useful info if you access to them:

Neubert, H. (1978) Vergiftung durch den Olbaumtrichterling,
Omphalotus olearius (DC. ex Fr.) Singer. Sudwestdeutshce
Pilzrundschau 14(1):20


Production of Antibiotics by Collybia nivalis, Omphalotus olearius, a Favolaschia and a Pterula Species on Natural Substrates
Key words: Basidiomycetes, Natural Substrates, Strobilurins, Oudemansin A, Pterulones
M. Engler, T. Anke and O. Sterner.     318-324
Z. Naturforsch. 35c, 1998

[Bubbleplate]

and got a copy of the original journal where Murrill posted his original discovery (Journal of the Florida Academy of Science Vol. 8, pages 180,181,and 198 year 1945) and there is no mention of alkaloid percentages. I also find curious the fact that after the Patent was issued, so far, I have not been able to find any mention of commercial production by any company. Perhaps the European Ergot producers (mostly in Switzerland, i.e. Sandoz) dropped the price on field grown Ergot in order to stifle competition.
Still, I feel that Omphalotus olearius presents a real opportunity for home grown Ergot Alkaloid production.
Perhaps there are even other mushroom fungi "out there" that can produce Ergot alkaloids! As far As I know, no one has looked to see if there are....

[Bubbleplate]

Patent is talking about. At the point in time that Murrill made his discovery, what we now call Omphalotus olearius was classified as Clitocybe illudens. So Murrill's designation of C. Subilludens was an attempt to say a "sub species" of illudens, hence the subilludens. I truly believe that Murrill just happened to find a unique local variation of the regular ol "Jack-O-Lantern". It is widespread not only in Florida, but most of the Eastern half of North America. Until people get out there and start testing, who knows how widespread Ergot alkaloid mushrooms really are!
I smell a great college research project and funding opportunity!

[bottleneck]

Incredible! But please could someone find a yield on these little buggers? I just hate patents where they don't give a yield.

I guess maybe one ought to learn a little about sterile technique...

[sean1234]

i agree, this could be huge if even the alkaloid content isnt as mush as that of claviceps...sounds as though the culture isnt hard to maintain at all, plus the
maybe a more industrious bee can go hunting, or maybe get into a mushroom cult ring and request that a sample be sent for trade. Too bad though that the mushroom itself is dependent on a host organism..i assume that it would be impossible to fruit to maturity "in captivity".
nice find!

[bottleneck]

We must find this mushroom! Gee, I can't believe I am going to have ask shroomers for help on making precursors...

Sean, what do you mean it depends on a host organism? In the patent, I think it just says something about growing it on normal liquid media. And it grows fast, so should be resistant to contamination!! Maybe a "beer-brewing" style set-up would actually be feasible for large quantities of broth? Hey, anybody got a couple of old bathtubs or maybe a spare swimmingpool? I suddenly find myself in need of a big container.

And with a basidiomycete like this, it should be very possible for people to do _selective breeding_. See a carp you like? Then print it and share! Muhahaha! It seems wrong and unjust that this mushroom should only be growing in Florida.

I don't think causing mutations will be needed to nurture the good qualities. Just look at the success of our brohers in the Cannabiz! To understand what breeding can do, just think of the Huxleys. What we need is the Huxley family equivalent of Clitocybe subilludens.

Hmm, which culture collection to order from... Or maybe just wait till summer and hope you come across something glowing in the dark.

Coincidentally, the patent also recommends extracting with active carbon, just as was suggested in this forum some days ago.

I wonder, if you add carbon to the broth while the mushroom is growing, and thereby "suck up" the alkaloids from the broth, will this "inspire" the mushroom to produce more alkaloids than it would otherwise have done? Could one thereby go a way towards achieving the 5 kg/mL yields I am hoping for?

The answer is yes! Probably. Why not? And if it doesn't, we'll teach it!

[sean1234]

I was assuming that the mushroom was like amanitas in that the mycelium can be cultivated but it cant be brought to fruit due to its sybiotic relationship with the tree, but i could be wrong....not that it really matters anyway unless the mature mushroom has more alkaloids than the mycelium, which it might.

I don't think causing mutations will be needed to nurture the good qualities. Just look at the success of our brohers in the Cannabiz!

this goes back to selective strain isolation on agar plates.

[Bubbleplate]

This mushroom grows just about ANYWHERE in the USA along the East Coast (Maine to Florida) where ever there are hardwood trees. And that's just where I can personally vouch to have been/seen them. I have no doubt in grows in most of Eastern Canada and probably further West in the USA.
Clitocybe mushrooms (and the actual fungi) are quite easy to grow in culture. Sterile medium and proper conditions (temp, O2, etc) and away you go. I have seen this species growing on rotten log debris in my backyard in clumps of 20,30 and more mushrooms. Very hardy fungus.
The trick is going to be finding that "one" that has Ergot alkaloids. Me, I'm going to buy a few "LSD" test kits (on sale) at the local Pharmacy, and come this Summer/Fall, start testing every clump I see. Once ONE of us finds the "magic" sub-species, we just pass the spores/cultures around and we're off to the races!

[Cept]

i thought that the jolly lolly floppers got shut down a while ago? they hooked up all sorts of shady shit like sassy oil, 2-CT-7, benzylpiperizine, lots of claviceps, fucking shit even human fetuses! i swear to god! the list goes on: bufo vulgaris poison toads (bufotenine[5OH-DMT]/5meo also probably), dream/puffer fishes,5MeO-DiPT!,that's all i remember but i mean, seriously they had to know it was coming, they're fucking shady as sin.

[bottleneck]

Sean:

>I was assuming that the mushroom was like amanitas in that the mycelium can be cultivated but it cant be brought to fruit due to its sybiotic relationship with the tree, but i could be wrong....not that it really matters anyway unless the mature mushroom has more alkaloids than the mycelium, which it might.

Oh, you are probably right about those difficulties. It would be nice to be able to get spores.

> this goes back to selective strain isolation on agar plates.


I guess? But at least when you can get spores, it should be easier to develope new varieties because you don't have to add mutagens and stuff like that.

Biojammer:

> spare swimmingpool? dear god, man!

On the other hand, if the mycelium needs a lot of oxygen, i suppose a jacuzzi might be handier.

About Erlich's reagent, I am not sure, but it sounds as if just about any indolic compound will react, including tryptophan. The standard colour is blue, I think, but there is a range of possible colours from pink to violet and even green, depending on the compound. Of course, a pure extract from a mushroom will most likely have a range of different compounds, and the combined colour of the mixture can't be used to identify the parts of the mixture.

I am not sure if there even is free tryptophan in any mushroom, but extracting the mushroom before testing with Erlich's reagent probably ensures no competing indolic compounds are present, as is stated in the following article referring to ergot:

Public Health Reports;1930;45;1466-81;Smith;A Quantitative Colorimetric Reaction for the Ergot Alkaloids and Its Application in the Chemical Standardization of Ergot Preparations.

(http://www.geocities.com/botero56/PublicHealthReports.1930.45.1466-81.Smith.pdf):

However, additional reliability could probably be gained from doing paper- or thin-layer chromatography on an extract of the mushroom, and then spraying the finished chromatogram with Keller's (Erlich's) reagent. That way, most bands will probably consist of unique compounds , so the colour given is accurate for the compound (as is kind of the point of chromatography, I guess). Also, the distance of each band has travelled in relation to eachother from the beginning spot will be pretty much identical on any chromatogram developed under identical conditions, and so can be used to identify the compound.

With the huge interest in psilocybin-containing mushrooms within mycology, it seems strange to me that mycologists wouldn't have already tested a lot of species of mushrooms for indolic compounds, so shouldn't they have already registered this Omphalotus as containing indoles?

What might have happened, though, is that thin-layer chromatography has been used to test them, and that they have only looked for psiloc(yb)in, and ergolene compounds are missed. It might be interesting to check out some monographs on Omphalotus to see if some references for chemical testing of the genus are given.

Still, if modern, hugely expensive testing technique like LC/MS has been used, they should have identified the ergolenes. There should really be articles on this already available, I think. A trip to the local Botanical Library, perhaps?

Edit: What I mean is something like this:

http://www.nationaalherbarium.nl/pubs/persoonia/pers17_4.htm

"Chemotaxonomical and morphological observations in the genus Omphalotus (Omphalotaceae)
Comparative thin-layer chromatography – for the first time applied to Omphalotus olivascens var. olivascens, O. olivascens var. indigo, O. nidiformis, and to O. mexicanus – revealed strikingly similar pigment patterns for all Omphalotus species except O. mexicanus. Atromentin, thelephoric acid and pulvinic acid derivates were found in dried material and/or culture extracts of all species. Illudin S and illudin M were detected in O. mexicanus by high-performance liquid chromatography-atmospheric pressure chemical ionisation mass spectrometry. Data on morphological features of all described Omphalotus and Lampteromyces species are listed, illustrated, and summarized in a key. New combinations in the genus Omphalotus are proposed for Lampteromyces japonicus and L. mangensis.

When they have examined these species with HPLC/MS, you can bet they have examined Omphalotus illudens and many other north american species. In fact, this article in Persoonia might be a good place to start for references for those examinations.

Many in the genus contain muscarine, so "hallucinations" should probably be qualified:

http://www.inchem.org/documents/pims/fungi/pimg028.htm

[Bubbleplate]

>>>have already tested a lot of species of mushrooms for indolic compounds, so shouldn't they have already registered this Omphalotus as containing indoles?<<<<

Well, that's what I was thinking, BUT, when I searched literature to see if anyone HAS looked for indolic compounds in mushrooms, I couldn't find any! Not to say no one has looked...but I haven't seen any such research.
Even the infamous Dr. Albert Hoffman, discoverer of LSD & psilocybin in shrooms, didn't believe his own data when when it showed that LSD like compounds were to be found in Morning Glory seeds! He initially thought it was "lab cross contamination"!!

DNA & genes, especially in the wild, has and will continue to give humans a surprise or two! DNA has a "mind of its own"...

I totally agree if that if one is going to look for indolic compounds in shrooms (or other "plants") one should do an extraction; Then dropping extracted solvents on chromatography paper, or blotter, and putting under UV light should be done.

[bottleneck]

> Well, that's what I was thinking, BUT, when I searched literature to see if anyone HAS looked for indolic compounds in mushrooms, I couldn't find any!

Well, I haven't been interested in mycology for a while, but I do have a slip of paper saying at least four species of Inocybe contain psilocybin, I. aeruginascens, corydalina, haemata and tricolor, and references for Bels & Mack, Z. Myk. 51, p. 183 (1985) and Gartz & Drewitz, same journal, page 199. Since Inocybe are supposedly closely related to Clitocybe, it would seem logical if most members of this genus have also been tested.

I am not sure, but I seem to remember there is a publication called Botanical Abstracts, serving a function like Chemical Abstracts, indexing the botanical journals. Maybe that would be a good place to start to find references for "chemotaxonomical" testing for indoles.

> Even the infamous Dr. Albert Hoffman, discoverer of LSD & psilocybin in shrooms, didn't believe his own data when when it showed that LSD like compounds were to be found in Morning Glory seeds! He initially thought it was "lab cross contamination"!!

I think you're right, there does seem to have be some controversy at the time. But since then, I think they have had plenty of time to digest the news, and must have probably tested almost everything living for ergolenes.

> DNA & genes, especially in the wild, has and will continue to give humans a surprise or two! DNA has a "mind of its own"...

You're probably right. But I believe HPLC/MS is very sensitive. Just about every peak on the chromatogram can be identified with a very high degree of certainty from either a database of spectra or by straight interpretation of the mass spectral data. They just have to inject a sample and press a button, and about 30 minutes later, a nicely formatted sheet of paper will list the quantities and identities of every molecular component found in the sample extract.

It could be interesting to check out a lot of articles on such tests of mushrooms, to see if any compounds which are known to act as ergolene precursors in lower fungi are present.

> Then dropping extracted solvents on chromatography paper, or blotter, and putting under UV light should be done.

Are lysergic acid amides fluorescent?


Biojammer:

> actually, i saw them growing on dead poplar tree stumps. that's where i've seen 99% of them anyway. it's not symbiotic, it's not even parastitic. the stumps were very dead already.

So, it's just a normal shroom. Good!

[foxy2]

But I believe HPLC/MS is very sensitive. Just about every peak on the chromatogram can be identified with a very high degree of certainty from either a database of spectra or by straight interpretation of the mass spectral data. They just have to inject a sample and press a button, and about 30 minutes later, a nicely formatted sheet of paper will list the quantities and identities of every molecular component found in the sample extract.

Uhh yea right, its obvious you haven't used one of them.  If you already know whats there and what your looking for its not to difficult, if not then well......

Ask someone who's an expert if you could just shoot a mix of unknown(possibly never before identified) ergot alkaloids into a HPLC/MS and get the result you stated.  Somehow I doubt it.

[bottleneck]

>Uhh yea right, its obvious you haven't used one of them.

Really? That is sharp of you. I was really trying so hard to give the impression that I had. That's why I used the words "I believe". Have you used one? Good for you.

Next time I'll be sure to write a 300-page essay and have it peer-reviewed before giving my opinion on something in your presence. But I'll have you know I'll expect real good payment from you.

Somebody pee on your sugarbread?

Don't you know nothing is real, least of all experts? Or have you been a wageslave for too long to remember?

[pashov]

The occurence of osytocic alkaloids in this mushroom has been further investigated in
TYLER V. E., 1961 - Indole derivatives in certain North American Mushrooms. Lloydia, 24: 71-74.

I can't get that... Maybe somebody else can?

[bottleneck]

Superbe!

I have ordered it, and I should receive and be able to scan it at latest next tuesday, I think. If someone can get it earlier, please post a link.

[bottleneck]

Searched for that article by Tyler. Found this large number of references:

http://www.samorini.net/doc/sam/basidio.htm

(Excuse my Google-italian):

"We remember finally the Clitocybe gallinacea Scop.  former Fr., rare in Italy, recovered as an example from the Saccardo in the province of Parma (1915), and in which v' she is who it thinks to have to you characterized alkaloid of lisergico type (cited in ARIETTI & T0MAÏ, 1975), made not little amazing since would be one of the little cases (the only one in the European micologia) of production of this type of alkaloid (well-known produced from the purpurea Claviceps, ergot) between the advanced fungi.

We remember, between the other, than also for the North American species Clitocybe subilludens Murr.  previously it had been asserted produced alkaloid of ergot (FOOTE ET al., 1953), made then refuted from studies more deepened (TYLER, 1961).  It is from holding pure in consideration that this fungus belongs to a kind of which much species, in particular the Clitocybe white women, is considered toxic since muscarinici compound manufacturers.

ARIETTI N. & TOMASI R., 1975 - I funghi velenosi. Bologna (Edagricole)."


Very confusing. Seems to say that the Tyler article refutes the claims of the patent describing production of lysergic acid amides by Clitocybe illudens, yet talks about some other claim of lysergic acid amides in gallinacea.