how to make paspalic acid from Claviceps paspali

[ben135]

hi i was wondering if anybody knows how to get paspalic acid from the Claviceps paspali fungi

thnx and greetzz ben

[Rhodium]

See the references in

https://www.thevespiary.org/rhodium/Rhodium/chemistry/lsd.synthesis.txt

- and try our fine search engine! It is in great shape, and as good as new - it is almost never used by people wanting to find information!

[ben135]

i used the search egine but did not find the formula for making paspalic acid just that it forms when you place claviceps paspali under saprophytic fermentation conditions
but i don`t know wat that means saprophytic fermentation conditions can some one help me on this

btw is it hard to get paspalic acid is it a controled substince or not i live in belgum do you think i can get it here tryed getting ergortamine tartrate but i coulnd get it without a prescription from a doctor is

[El_Zorro]

You might start my looking at the links in this post:

Post 394021

(El_Zorro: "From Dallis grass to lysergic acid (in theory)", Tryptamine Chemistry).

[Bubbleplate]

in a liquid medium under sterile conditions to get any decent yield.
However, before even doing that, you need to isolate a strain of C. Paspali that produces alkaloids in decent quantities first. This can be done on agar plates and then scaled up to liquid medium. (Unless of course you already have access to a strong alkloid producing strain). Some strains will produce Lysergic Acid Hydroxyethylamide, D-Lysergic Acid Amide, and D-Isolysergic Acid as the main product.
If there is any real interest, I will post details about the liquid culture medium and such.

[ben135]

i can oder the c. paspali strain here

http://www.dsmz.de/species/sp300279.htm

but i can choose out of 4 different strains can you tel me witch one is the best for paspalic acid production
also how do you get paspalic acid from the strains
 i read somewhere that the fungi produces paspalic acid in the medium and that i can extract it from the medium  using the a/b methode but i don`t know what that methode is can some one tel me of this extration is done  also dos the fungi alway produce the acid or only under certen conditions if so wat are these condistions
Bubbleplate i am very interrestid i the liquid culture medium  that is used to grow the fungi so that it produces high yields of paspalic acid

thnx and greetz ben

[Yachaj]

Post No 340178

Inoculate halfpint container which contains 100ml of solid peroxidated maltose/marmite gelatin with bit of fungus (h2o2 is optional but enables desktop inoculation/incubation without hepa). The surface will be colonized in just a few days, then the solid gelatin liquefies. Remove fungus (paspalic is in the medium). Add HCl. Do A/B. Save crystals. Expect a yield of 100mg of crude product per 100ml of gelatin.

Do not ingest.

[Bubbleplate]

will do, but if it were me, I'd go with the 884 or 885.
What is your focus on Paspalic Acid? Those strains you're looking at will produce Lysergic Acid Amide derivatives.
Alkaline hydrolysis of those and you get lysergic acid.
What is it you're trying to do? If your goal is to get a decent amount of lysergic acid, then liquid fermentation is the way to go. With a good strain one can get about 1 gram per liter of medium. Check those Patents referenced at the culture collection.
Word of advice: if you haven't done sterile culture before, you will want to practice on something easier/safer than Claviceps. Maybe practice with psilocybin fungus first.

[ben135]

so strain nr 884 and 885 pruduce l.s.a derivatives and not paspalic acid if so do i extrack it the same way from the medium as paspalic acid and can you tel me wath they mean with a/b extraction methode
p.s
 i have never done sterile cultere but i`l think i can menage it

[Lilienthal]

Please use the search engine, the links therein, or the Abbreviations page (Hive navigation bar).

[ben135]

i searcht with the search engine but i stil can`t find a answer to my questions let me ask my quetions ons more

1 how dos the a/b extraction for extraction of paspalic acid from the medium where the c.paspali fungus is cutuvated in work (what chimicals do i used and how mutch of them do i use in for example 1000 ml of medium )

2 whats are the best chimicals to make the medium with and whats the best way to keep them sterile i was planing to use to set up given in the book acid trips and chemistry by sam cloud in the book they cultuvate c.purpurea but i ques the way of cultuvating is the same only the medium would be different

can someone give an anser on these questions please or give direct links or pattents on where i kind find the correct answer to these specifick questions (for give my englishe its because i`m from holland )

[Bubbleplate]

On a scale of 1 to 10, growing Claviceps AND getting anything out of it is an 11!
Forget about growing C. Purpurea - it will make tons of fungi, but you will NEVER get any product from it. Trust me on that one!
I still say you need to practice Sterile culture first. Try making medium and isolating ONE fungi strain (mushrooms, bread mold, whatever) and once you get good at that, move up to Claviceps.
Here's the formulas the Italian scientist who isolated C. pasapli first used:
1) Agar plates:  Glucose-potato agar medium - glucose 20 gm., potato infusion 300 gm., agar 15 gm., h2O 1 liter, adjust to pH 7. Grow out C. paspali strains and test different strains for maximum alkaloid yield with Ehrlich-van Urk reagent.(p-di-methylaminobenzaldehyde in 65% H2SO4)
Look for strains that produce Purple mycelium as these will have the highest alkaloid yield. Grow for 5-10 days and then move to Shake Flasks.

2) Once you have a good strain, scale up to Shake Flasks using this liquid medium: Mannitol 4%; Succinic Acid 1%, neutralized to pH 5.2 with ammonia; Chick Pea Meal 0.1%; KH2PO4 0.1%; MgSO4.7H2O 0.03%; H2O 1 liter. (all % by weight)
Shake Flask table should be run at 220 revolutions per minute, eccentric throw 10 cm, at 24 degrees C. Use 500 ml Erlenmeyer flasks with 100 ml medium.  Innoculate with Agar plates/slants grown for 5-10 days, then homogenized in Waring Blender with liquid medium under sterile conditions. Grow shake flasks for 4 to 5 days and then use to innoculate Fermenters.

3) Fermenter Medium: Mannitol 5%; Succinic Acid 3%, neutralized to pH 5.2 with ammonia; KH2PO4 0.1%; MgSO4.7H2O 0.03%; H2O 1 liter. (all % by weight). Fermenter vessels should have form ratio of height to diameter of at least 3 to 1 (i.e. height of vessel 3 times its width) Stirring is required from beginning by using "turbine propeller" type top-driven stirrer. Put Shake flask culture in waring blender and innoculate Fermenter vessel. Growth of culture should be at 24 degrees C. After 6 or 7 days, aeration needs to be increased by pumping sterile air at pressure of 1.4 atmospheres at rate to bring Oxygen level up to 70-80% saturation. Let culture grow no more than 8 or 9 days.

After 9 days, filter culture medium through large Buchner funnel or filter press. Adjust filtrate to pH 8, and extract with equal volume of chloroform/isobutanol mixture (4:1).
It helps if one has a centrifuge if large amounts of culture medium are being processed; The solvent phase is separated and then the soluable alkaloids transfered to water by shaking three times with 1/10 volume of H2O, adjusting the pH to 3.5 with sufuric acid. About 75-80% of the lysergic acid derivatives will be in the aqueous phase. The aqueous extracts are brought to pH 8 with dilute soda, and after addition of 1 mg. Versene (preservative) for each 10 to 15 mg. Lysergic acid derivative, extracted 3 times with half a volume of chloroform. Then the chloroform extract is evaporated in vaco to 1/50 or 1/00th of its volume IN THE DARK. The Lysergic acid derivitives will crystallize out. The remaining LAD's can be precipitated by adding petroleum ether, or by adding ethereal maleic acid to recover as maleate. Please note that LAD's are extremely sensitive to light. Lab work should be done under red light (darkroom) conditions!

[Bubbleplate]

are from the lab notes of the people who discovered the Claviceps Paspali strain that produces appreciable amounts of lysergic acid derivatives. This work was done in the late 1950's and early 60's by F. Arcamone, E.B. Chain, A. Tonolo, Lidia Vero, A. Ferretti and others at the Center for Chemical Microbiology in Rome Italy.
Yes the ORIGINAL strain of Claviceps paspali was "wild". After years of unsuccessfuly trying to get Claviceps pupurea, Claviceps litoralis, and other strains to produce any appreciable amounts of lysergic acid compounds in submerged culture, they decided to look at in greater detail the mechanism of sclerotia formation in vivo. For this purpose rye embryos (grass seed) were infected with different strains of Claviceps and then grown on agar. Under these conditions, some Claviceps strains were non-infective, some caused infections but grew in the form of vegetative mycelium without sclerotia formation, and a few strains gave rise to both infection AND sclerotia formation in the plant. One strain in particular was isolated from a sclerotium found on an infected plant of Paspalum distichum on a hill in Rome, and was identified as Claviceps paspali (Steven & Hall). A sub-strain, identified as F-140, was 90% effective in giving rise to sclerotia.
This strain was further improved by selection. It was seen that 3 types/colors of mycelium formed IN SUBMERGED CULTURE: White (28%) Brown (69%) and Violet (3%). ONLY the Violet colored strains produced Lysergic acid in culture. Also, colonies grown on Agar alone did NOT show any marked differences in color! Only when grown in liquid SUBMERGED culture.
 These violet strains were further isolated and selected for Lysergic Acid production (by testing cultures with Ehrlich-van Urk reagent and chromographic spotting under UV light)Speaking of UV light, these researchers used high intensity UV light to cause mutations in Claviceps paspali strains. It was found that by irradiating cultures with Long & short wave UV light, MOST of the fungi would be sterilized & killed. However, some of the few that did survive were found to have much more production of Lysergic Acid compounds. A few of the patents egarding C. paspali make off hand reference to this fact.
One strain, F-550, was chosen for large scale testing/production in submerged culture. (See my first post for methods) F-550 and similar strains could produce about 700-1000 micrograms of Lysergic acid a-hydroxyethylamide per ml of culture after only 6 to 9 days growth! Thats about 1 gram per liter. By tweaking various parameters such as % of various chemicals in the culture media, temperature, pH, etc. yields were even higher, at about 1,200 - 1,500 ug. per ml.

[Bubbleplate]

would produce. The discovery, or more accurately, the "creation" of the original Claviceps paspali strain F-550, that DID produce Lysergic compounds was not an easy task. Apparently, ONLY Claviceps strains that form sclerotia in plants, and "Sclerotia like" formations in culture (Tonolo calls them "pseudoparenchyma" in surface agar culture, and "synnemata" [singular] in submerged culture) will produce ANY Lysergic A. compounds. They used the the method of infecting/growing out rye seeds just to find suitable strains to start the process. Then they used the time honored techniques of strain isolation/testing and mutation to develop ever more "potent" strains. The UV (black) light they used to mutate the strains is NOT the type one uses to light up their Day-Glo posters, but rather the type that is found in germicidal lamps, and some PROM erasers:

http://student.ccbc.cc.md.us/biotutorials/protsyn/mutate.html

http://plato.acadiau.ca/courses/biol/Microbiology/mutation.htm

Keep in mind that UV light is not the only thing that can induce mutations in living organisms. Chemicals, heat, cold, and other radiation like X-Rays, and Beta and Gamma rays can be used.  One technique is to expose fungi to levels of the mutagen that kills 99.99% of them. The .01% that survive have a mutation that hopefully not only helped it survive, but just MIGHT also be linked to more Lysergic production. There are other techniques and there are whole books on the subject.

Could one get lucky and just happen to stumble on a wild strain that also happens to grow well in culture AND produce good quantities of Lysergic Acid compounds? Possible, but doubtful. More likely is that you'd have to follow the same steps Tonolo et al did and patiently isolate, grow, test, and mutate many wild strains before finding a "good" one.
A better path would be to get hold of a known L.A. producing strain and take it from there. You'd get decent yields and you could then do your own mutation experiments to try and grow a "super strain".

[Bubbleplate]

Ehrlich-van Urk reagent, or similar test (OTC LSD test kit?) to see if strains are making Lysergic Acid compounds or just producing Red & Purple Pigments. USUALLY purple pigments are sign of LA production, but I'd make sure...
BTW, are those C. paspali strains, or some other?
Anyway, GOOD WORK!
Now get that glass-lined 50 gallon water tank ready!

[Lilienthal]

UTFSE about the color reagents.