This patch of grass was grown out from 2-3 wild collected plants for 4 years. Plants clipped to 3" at the end of October from fully mature plants. Samples were taken of the re-growth at the end of November/beginning of December. Green growing portions only despite some frost in the past month.

Plates were run on two separate extractions (PH1 and PH2) with the following procedure:

5-10 gm refluxed in MeOH for 1 hour (x2)
Made acidic (pH3) with dilute H2SO4 and de-fatted with CHCl3 (x3)
Made basic with 10% NH4OH (pH12)
Extracted with CHCl3 (x3)
Organic layers combined and evaporated to yellow brown gum
Gum dissolved in 2ml MeOH and used for plates

Plate 1 system: IPA:Ethyl Acetate:NH4OH (60:15:5) Viz: .1% Ethnolic Xanthydrol with Phosphoric Acid
Plate 1, lane 1 - shows the MeOH only extraction from the beginning of the above procedure
Shows 5-6 compounds and that these are 'M' type plants. Beginning of extraction a little dilute for much more data
Plate 1, lane 2 - MHRB comparison for 'T' type determination (Control)
Plate 1, lane 3 - PH1 Extraction - unsuccessful extraction final solution too dilute. Does show 'M' type plants

Plate 2 system: IPA:Ethyl Acetate:NH4OH (60:15:5) Viz: 1% Ethnolic Xanthydrol with Phosphoric Acid

Plate 2, lane 1 - MHRB Control
Plate 2, lane 2 - Unidentified Virola sp. bark - extraction irrelevant, was hoping for 5-MeO in the bark and not found
Plate 2, lane 3 - PH2 full extraction, resolving 3 compounds. 2 with xanthydrol, one with fluorescence (Red) at a high Rf.

Plate 3 system: n-Butanol:Acetic Acid:H2O (12:3:5) Viz: 1% Ethnolic Xanthydrol with HCl

Plate 3, lane 1 - MHRB control
Plate 3, lane 2 - Virola - irrelevant
Plate 3, lane 3 - PH2 full extraction, resolving at least 8 compounds with the highest Rf circled showing a red spot under longwave UV before Viz.

Edit (see post below):

The most abundant compound in these extractions (PH1 and PH2) appears to be 2-Me-6MeO-1,2,3,4,THBC indicated by the largest blue spot at the lower/mid Rfs, showing similar relative placement on Plate 3 with comparison to the data in Gander et al (1976). This would also correspond with Woods and Clark (1971) who found that "The free growth grass produced at best a faint trace of compounds in the T or DMeT regions, but the compounds in the B-Carboline region rose steadily throughout the season". Table 2 also shows that the clipped plants produced more of the B-carbolines than compared to the free growth plants and that the Tryptamine compounds slowly declined through the season after peak production.The other trace spots show 5-MeO with what are probably residual trace compounds in the bio-synthesis of 5-MeO and 2-Me-6MeO-1,2,3,4,THBC.

The unexpected spot is the high Rf spot that glows Red under UV (top circle in Plate 3, lane3). It appears larger than the other trace spots and shows up in both Plate 2 and Plate 3. I've not run across a reference to anything that glows Red in UV with regard to P. arundinacea.

Also note that n-Butanol:AcOH:H2O was a better eluent for resolving compounds in Phalaris (on Silica Gel F 200 microns) and that Xanthydrol in HCL showed better colorimetric resolution for substituted and non-substituted compounds.

-D.

Thanks a lot for the work! This is very interesting! So as I suspected before, this is another example of how wild grass is most likely not the best alternative source for DMT/5-MeO.

In any case Ill probably come back to this and ask some more questions

..great work, yeah thank you for the data Dozuki..

i wonder how much TLC work has been done on 'established' strains like AQ1?

I wouldn't be surprised to find results similar to these from any of the 'cultivated' varieties of Phalaris a. I just think its a good example of the variation in compound production based on the habitat, growing condition, time of year and method of harvest.

More Preliminary results:

The photo below shows Plate 3 from above and a new plate I will call Plate 4.

~ 1ml Naptha was added to the 2ml MeOH that was used to dissolve the Gum from the above extraction.
This was put on a 55 C water bath for 2 hours with intermittent agitation of the solutions
It was then put aside over night
The MeOH layer was pipetted off the Naptha layer and put into a separate tube.
Each of these layers were spotted on Plate 4:

Plate 4 system: n-Butanol:Acetic Acid:H2O (12:3:5) Viz: .1% Ethnolic Xanthydrol with HCl

Plate 4, lane 1 - MHRB control
Plate 4, lane 2 - Methanol layer
Plate 4, lane 3 - Naptha layer


This plate would seem to indicate that the large spot is not in fact 2-Me-6-MeO-1,2,3,4-THBC and is 5-MeO. 5-MeO is known to be slightly soluble in Naptha, the solubility of the Beta-carboline in Naptha is not know. This plate indicates that the large spot is soluble in Naptha and shows no other trace spots, even under UV. It does show the large spot that fluoresces red under UV. I suspect that this is a compound associated with chlorophyll as it in the same Rf that chlorophyll shows up in Plate one.

I need to run another extraction to find out if the trace spots in fact stay in the Aqueous layer when Naptha is used as the solvent. I don't feel that this preliminary test, with just a single run of Naptha is conclusive, but it seems intriguing.

I ran this plate in a different system and got the same results.

-D.

This image is Plate #2 from above and then a new plate #5 Viz. with Ehrlich's reagent and then pre-viz under longwave UV (Purple Plate).

This gives further evidence that my initial thoughts about the large spots being a beta-carboline are incorrect and is 5-MeO. According to Frahn and O'Keefe (1971) and Agurell et al. (1969) 6MeO2MeTHBC and 2MeTHBC do not visualize with Ehrlich's reagent. The 2 large spots do show up, however, the small spot in Plate 2, Lane 3 with a similar rF as the MHRB Control does not show up in Plate 5. I now suspect that this is 6MeO2MeTHBC.

After 10 years, finally running plates again on P. arundinacea. Back then I hadn't found a Gramine phenotype plant, but have now.

Here is a test plate with:

Lane 1: MHRB extraction for a 'T' type control (Purple)
Lane 2: 'G' Type plant (Gramine) (Pink)
Lane 3: 'M' Type plant (5MeO) (Blue)

Plate is run in BAW (BuOH:AcOH:H2O)

Thought I'd post this for an example of the Xanthydrol reaction for all 3 compounds. Colors are easier to discern in the actual plate. This is a scan of the plate showing a closer color of the plate than a photo.

-D.