Research The nexian phalaris breeding programme

[Grasshoppers]

Today, we conducted further Thin-Layer Chromatography (TLC) analysis on specimens of wild Phalaris aquatica and Phalaris canariensis. The results from the Phalaris aquatica samples revealed the presence of N,N-DMT and 5-MeO-DMT within the expected range, consistent with our previous observations of this particular wild accession.

However, the findings from the Phalaris canariensis samples were unexpected. The TLC spot corresponding to DMT retention factor (RF) did not exhibit the characteristic patterns associated with N,N-DMT, 5-MeO-DMT, or 5HO-DMT, leaving us puzzled at this juncture.

The TLC plate depicting these results is presented below:

 

[Sidisheikh.mehriz]

Today, we conducted further Thin-Layer Chromatography (TLC) analysis on specimens of wild Phalaris aquatica and Phalaris canariensis. The results from the Phalaris aquatica samples revealed the presence of N,N-DMT and 5-MeO-DMT within the expected range, consistent with our previous observations of this particular wild accession.

However, the findings from the Phalaris canariensis samples were unexpected. The TLC spot corresponding to DMT retention factor (RF) did not exhibit the characteristic patterns associated with N,N-DMT, 5-MeO-DMT, or 5HO-DMT, leaving us puzzled at this juncture.

The TLC plate depicting these results is presented below:View attachment 97943

Ok that's really strange. Those canariensis spots don't seem to correlate to any of the colour screening of the tryptamines standards we have tested. It can't be nmt either as it should have lower RF than DMT.

Only one way to find out. Bioassay!

By the way tyramine alkaloids may not be carried up in an acid/base extraction as they exhibit solutbility in water under alkaline conditions. Anderton, et al 1999. (Assessment of Potential for Toxicity of Phalaris spp. via Alkaloid Content Determination: P. coerulescens, a Case Example).

Quote: "Solid phase extraction (SPE) techniques using sulphonic acid cation exchange columns have been used to process plant extracts in order to detect the
water soluble tyramines that might be discarded in the usual acid/base liquid extraction procedure for alkaloids"

 

[Sidisheikh.mehriz]

Yesterday I performed a successful extraction on yet another harvest batch. These are mature seedlings from the cultivar parent clone, grown on well drained, saline lime soil. The seedlings have already flowered and 50% of the panicles have already dried and shed seeds. Majority of the biomass were in stems. Leaves were few and at the end of their life, wilting. These are plants that were in the transition phase into summer dormancy.

This is yet another result that contradicts the agronomic literature on aquatica. Alkaloids according to CSIRO are highest during autumn and gradually decrease to the lowest concentration in spring after anthesis.

My yield from this late spring extraction was actually quiet decent! I had similar results from last year as well with the parent clones. So clones just as the seeds from the clone both shows similar alkaloid profile and both yield well in late spring. Guess the literature isn't all that accurate after all.

Bioassays are coming today.

 

[donfoolio]

I think that besides the season of the year, growth stimulation might play a more influencal role.
There is a huge difference between wild growing poacacae and plants that have been pushed to regrowth through grazing, cutting or other forms of cultivation.

This might be the more important factor.

Keep up the good work!

 

[Sidisheikh.mehriz]

Just did a couple light hits. It's definitely not 5-meo-dmt. It's something else that doesn't cause fear and panic. Smooth,, relaxing, euphoric and definitely psychedelic in a tryptamine way. Colours are saturated at this light dose. Enjoyable tingly sensation on my skin. Need to plunge a little deeper. I'll keep editing as I smoke more.

 

[Sidisheikh.mehriz]

I think that besides the season of the year, growth stimulation might play a more influencal role.
There is a huge difference between wild growing poacacae and plants that have been pushed to regrowth through grazing, cutting or other forms of cultivation.

This might be the more important factor.

Keep up the good work!

Wild harvested phalaris of all sorts of species have proven active as well and some with pretty good yields. So the harvesting regimen doesn't necessarily play a major role. For cultivated aq in my experience as long as I keep the grass short it always delivers a good yield. 300 to 600g fresh is good range. I saved leaves from today's batch for analysis.. grasshoppers will TLC it see what it has to understand what to look for in other strains to get similar extract effects. This is the kind of extract you can smoke casually like weed.. its pretty chill and soothing. Having some difficulty vaping it properly so far and it may prove to be potently psychedelic once vaped correctly at sufficient dose. I love it

Edit: did a higher dose and it's gotten more visual with closed and open eyes flashy colours and morphing objects. Starting to feel like DMT.

 

[Grasshoppers]

For improving plate comparability, we employed an new approach by quantifying the integral of spot darkness along the height profile of a specific substance. A custom script using OpenCV was developed and applied to analyze a dilution series of caffeine, with a consistent dilution factor of 2, as a means to validate this methodology.



The results of our experimentation, illustrated in the image below, demonstrate the relationship between the measured integral values and the concentration of caffeine. Although the observed correlation is not strictly linear, it remains sufficiently robust for potency assessments and facilitates comparison of substance concentrations.



However, it is imperative to acknowledge a notable challenge encountered in our analysis — the inherent sensitivity of Phalaris to environmental variables. The considerable phenotypic variability observed can significantly impede our ability to elucidate the underlying genotype.

Addressing this challenge represents a current focal point of our research efforts. We are actively exploring strategies to mitigate the influence of environmental factors and enhance the reliability of our comparative analyses.

 

[Sidisheikh.mehriz]

Awesome work grasshoppers. I hope we can build a similar graph with nn/5m standards for more practicality. This may prove to be a reliable quantification method.

In light of my most recent bioassay I'm obliged to question yet again my long held belief that this is a completely stable profile cultivar. The alkaloid profile variability in the span of three years seems rare and sporadic however. Three years of cultivation and bioassays shown 5m like effects to be the major character with non 5m extracts expressed only twice during those three years. That's not a substantial degree of variability but it's still something worth noting.

Last night I did even a larger smoked dose using Ziziphus lotus leaves and paganum harmala flower petals as the substrate. It lead to highly visual near breakthrough experience. Unlike 5-meo-dmt effects from previous extracts there was no panic, no loss of sense of self instead my visual field was morphing then dissolving into a mush. Like a painting washing away in the rain. Room started to morph then objects contours have dissolved and merged with each other and so was my thoughts. It's. Kind of like Albert Hoffman LSD bycicle trip. There was no cardiovascular activity, zero nausea, less body centered effects. All cues were suggesting DMT as the main character of the extract. Smoking same extract using only harmala flowers without ziziphus lotus leaves was a lot milder and less visual this lead me to think something in ziziphus (commonly known as sidr in the middle East) contributes to the effects. I smoked sidr leaves alone and it had a relaxing soothing effect but no psychedelic effects of its own. Harmala flowers smoked alone gave flashy visual field and effects similar to smoked hamala freebase. The synergy between all these three gave way to the most colourful and balanced experience both quality and potency wise.

I wrote in a previous post in this thread

I previously talked about seasonal variation in alkaloid profile but later discovered that the perceived profile variability was merely altered smoked experience effects due to varying dosage along with ratio of tabacco to extract in a joint (tabacco alters the experience whether due to nicotine or from harmane content. Black tea as inert base didn't alter the experience in comparison) this has become even clearer to me once I started vaping extracts Instead of smoking. 5-meo-dmt which am sure is the main alkaloid in my aquatica is known to bring very different effects depending on dose. It can feel completely like a different substance altogether. So yeah I will have to bring this up again in other threads and correct that info.

that what I thought was variability in alkaloids profile in winter of 2022 was merely subjective effect from light dose of 5m extract..but I can confirm now that I had that same variability with last night extract as in winter of 2022 but managed to dose higher and delve deeper into it till I fully understood it to 100% confirm it's not 5-meo-dmt.

Here's that bioassay report from 2022 winter. You can see the similarities in that report to last night's report.

To sum it all up. Clones if this aquatica cultivar still mainly have a stable 5m profile with some sporadic DMT like harvests (only twice in three years). This sporadic shift in alkaloid profile was observed in both the parent clone as in the seedlings from the clone. Seedlings also exhibited mainly 5m character throughout the growing season (3 bioassays from three harvests across the span of the growing season from small seedlings to end of growing season) with only last harvest "4th harvest" expressing DMT like effects.

Do I regret this variability? Absolutely not, i like good surprises hahaha. This is a local cultivar that's been bred for 10 years and is reported to be 98% genetically stable according to our national gene bank. I think that phenotypic expression of a given genotype (response to external factors) can further complicate things for this breeding programme but the main character of a given genotype practically should still be possible to preserve through selective breeding. My take away conclusion from these three years of experimentation with just one cultivar is that variability from phenotypic expressions in a cultivar are more pronounced than genotypic variability.

Now that I have confirmed that this most recent harvest batch contains no 5-meo-dmt my next step is to try an Ayahuasca analogue from the same batch and report back.

 

[dithyramb]

Cool news. IME "Anomalies" in alkaloid profile within stable single strains of poaceae are linked with seasonal and environmental conditions and stage in life cycle. Let's try to pin down the associated patterns (disclaimer... İt takes years.)

Eagerly waiting for the aya analogue report. IME even with lack of 5, aquatica is more "paternal" than brachystachys, the visuals are less morphing/speedy, and more glass or "oil painting" like. İf your experience has no side effects or unhealthy feel, this alkaloidal state of aquatica would be worth pursuing as a counterpart for rue.

 

[Sidisheikh.mehriz]

Cool news. IME "Anomalies" in alkaloid profile within stable single strains of poaceae are linked with seasonal and environmental conditions and stage in life cycle. Let's try to pin down the associated patterns (disclaimer... İt takes years.)

Eagerly waiting for the aya analogue report. IME even with lack of 5, aquatica is more "paternal" than brachystachys, the visuals are less morphing/speedy, and more glass or "oil painting" like. İf your experience has no side effects or unhealthy feel, this alkaloidal state of aquatica would be worth pursuing as a counterpart for rue.

I can't say it's a seasonal variation as this theory didn't hold up in my experience. I previously held the belief that non 5m profiles are expressed seasonally in cooler winter temperatures and hypothesised that enzymes relating to tryptamines synthesis exhibiting lower function at colder temperatures but that theory didn't hold weight when I had same extract in late spring. Yet previous spring this milder extract was non existent neither in late spring nor in winter. My experiments show this variation to be rather rare and sporadic.

I have high hopes for an Ayahuasca analogue with this cultivar from this specific harvest batch. Most likely the perceived toxicity of aquatica is merely the side effects of 5m with maoi and is dose dependent. I would love to be proved wrong but I've yet to see any reports/experiments to consolidate such seasonal variability in aq.

 

[dithyramb]

I can't say it's a seasonal variation as this theory didn't hold up in my experience. I previously held the belief that non 5m profiles are expressed seasonally in cooler winter temperatures and hypothesised that enzymes relating to tryptamines synthesis exhibiting lower function at colder temperatures but that theory didn't hold weight when I had same extract in late spring. Yet previous spring this milder extract was non existent neither in late spring nor in winter. My experiments show this variation to be rather rare and sporadic.

I have high hopes for an Ayahuasca analogue with this cultivar from this specific harvest batch. Most likely the perceived toxicity of aquatica is merely the side effects of 5m with maoi and is dose dependent. I would love to be proved wrong but I've yet to see any reports/experiments to consolidate such seasonal variability in aq.

The cultivar you are working with is indeed a result of the interbreeding of various different wild strains, so that would make different genetic expressions showing up not surprising.

About side effects in combination with rue, my experiences with the DMT dominant and most likely 5 MeO DMT lacking (but of course needs analysis to say for sure) aquatica gave me unpleasant physical stimulation in the legs during the experience and afterglow (in the extreme case, as I have mentioned numerous times, it caused heart overstimulation and a feeling of physically being on the verge of death. That strain did not have 5). whereas with the clearly 5 dominant strain this side effect was milder, but that one had a consciousness suppressing side effect to it. I am pretty certain aquatica has a very complex psychoactive content and that the effects and side effects are beyond DMT and 5 MeO DMT.

5 MeO DMT is not known to be a physical stimulant. Tyramines are. And what causes the consciousness suppression, we have no clue. This side effect is noted in some other aquatica and rue experience reports as well.

Stay safe.

 

[Sidisheikh.mehriz]

A honey bee landing some help

 

[Sidisheikh.mehriz]

The ayhuasca worked strikingly well! Perfect synergy with rue. One and a half table spoon of rue cooked for 40 mins drank all at once. 40 mins later drank 10ml AQ tea worth of 1g dry. I hit the third hour mark and it's still going. At this dose it's mildly visual. I have to unfocus my gaze on an object for it to begin morphing but moving around makes it more intense. Feels nothing like 5-meo-dmt so my guess was right when smoking it. Almost no nausea, feeling energized and light.

It started very mild on the verge of placebo then it started coming down and peaked again. Leaves were sun dried then blown with a hair dryer till it's fully crisp. Powderized and cooked with a pinch of citric acid for 1h just once. I did 100g batch and used only 1g

 

[Grasshoppers]

Intriguing discussion, @Sidisheikh.mehriz and @dithyramb!
To comprehensively address the variability of alkaloids across seasons, we will have to conduct periodical testing over at least one year on carefully chosen mature plants. This extensive duration will allow us to capture the nuanced fluctuations in alkaloid levels within the selected plants, thereby enhancing our understanding of their dynamic nature.

@Sidisheikh.mehriz, your contribution through bioassay methodologies is greatly appreciated, further enriching our understanding in this domain.

Presented below is another TLC analysis showcasing additional clones primed for assessment in our breeding program.


All the tested plants exhibit 5-MeO-DMT. Building upon our earlier identification of numerous plants within the U accession, we'll utilize the Sa accession specimen to bolster genetic diversity within our breeding line.

 

[Grasshoppers]

Today marks a notable milestone as we unveil the TLC results of the Tanit accession, a subject of considerable attention due to @Sidisheikh.mehriz's rigorous bioassay reports.

The Tanit sample is juxtaposed with samples from the U accession, a group we are diligently scrutinizing for promising specimens, given their recognized high yield.

Our findings validate our hypothesis of the Tanit accession being 5-MeO-DMT dominant. The distinct spot observed on the TLC plates serves as a benchmark for the yield requisite for personal use. Particularly noteworthy is the remarkably clean profile exhibited by the Tanit accession, as evidenced in the accompanying image.



It was surprising to find that the bioassay-validated Tanit accession is yielding considerably lower compared to several other specimens already identified for breeding. This disparity may arise from the relatively juvenile state of most other tested specimens, but it also suggests the potential for achieving significantly higher yields than those currently deemed necessary for extraction purposes.

At this point, it's crucial to discourage any attempts at bioassays with unknown accessions. Phalaris presents significant risks, especially given the high pharmacological potency of 5-MeO-DMT. Ingesting Phalaris without proper testing and expertise can lead to severe overdoses of various tryptamines and tyramines, potentially resulting in medical emergencies.

That being said, with a clean profile and safety validated through bioassays, we're thrilled to announce our readiness to distribute 5-MeO-DMT dominant Tanit seeds for the first time.

However, it's important to recognize that variations in environmental conditions may affect yield, and we have yet to assess the variability among individual Tanit plants. Understanding the precise environmental factors conducive to high yield will be the focus of our upcoming research endeavors, though this process is expected to take some time.

Initially, distribution will be restricted to esteemed members of the Nexus due to safety concerns. Clones will become available to the broader community without restrictions at a later date.

 

[Transform]

Quick question, since the major spots of the U accessions seem to be between those of N,N-DMT and the 5-MeO of the Tanit accession Rf-wise, have you tested any known mixtures of alkaloids for variations in separation resulting from, for example, association between the two very similar molecules leading to a kind of "braking" effect on the leading spot? Is there a post where you've shown some validations of separation (or maybe it was in the PDF and I've simply forgotten about it)? [Time for me to play the 'old' card]

 

[Grasshoppers]

@Transform
Previously we delved into the characterization of standards of N,N-DMT, 5-MeO-DMT and 5-HO-DMT, both individually and in combination. You can check out the outcomes in this post. Despite experimentation, utilizing 10cm 60Å standard silica plates in conjunction with various eluent systems, achieving adequate separation remained elusive. Intriguingly, the RF values remained consistent across the board, whether these substances were examined in isolation or in combination.

An observation emerged concerning Phalaris samples, revealing a variable, occasionally pronounced retardation effect. Despite performing through defatting using petrol ether, only partial alleviation of this issue was achieved, with complete resolution proving elusive, as highlighted in the aforementioned discussion.

Currently, digital post-processing of photographs captured from both wet and dry plates stands as the favored approach for discerning DMT-derivatives.

 

[Transform]

@Transform
Previously we delved into the characterization of standards of N,N-DMT, 5-MeO-DMT and 5-HO-DMT, both individually and in combination. You can check out the outcomes in this post. Despite experimentation, utilizing 10cm 60Å standard silica plates in conjunction with various eluent systems, achieving adequate separation remained elusive. Intriguingly, the RF values remained consistent across the board, whether these substances were examined in isolation or in combination.

An observation emerged concerning Phalaris samples, revealing a variable, occasionally pronounced retardation effect. Despite performing through defatting using petrol ether, only partial alleviation of this issue was achieved, with complete resolution proving elusive, as highlighted in the aforementioned discussion.

Currently, digital post-processing of photographs captured from both wet and dry plates stands as the favored approach for discerning DMT-derivatives.

Thanks for the further explanation, much appreciated. I do recall you having discussed the retardation effect now that you mention it - the precise terminology escaped me at the time of posting, possibly for the same reason that I wasn't up to tracking down the post which you have just, most kindly, linked to there. It does leave me wondering if there is any room for further improvement of the TLC separation process.

Besides the possibility of using pH to isolate 5-OH-DMT on account of its acidic proton, the only other major change that springs to mind would be 2-D TLC, but this has the disadvantage of only allowing one sample per plate.

 

[Grasshoppers]

Addressing the variability in retardation presents an opportunity for enhancement. Exploring improved defatting methods holds promise in this regard.

However, a critical consideration arises: which solvents are optimal for selectively removing more polar constituents such as resins while preserving the integrity of alkaloid salts within the sample?

The utility of TLC is undeniable, offering rapid phenotyping at a super cost-efficient scale. Yet, for in-depth analyses of specific specimens, transitioning to HPLC appears prudent to unlock richer data insights.

Thus far, our analyses have predominantly focused on P. aquatica and P. arundinacea, yet we have not observed any 5-HO-DMT presence. This suggests that this compound may not be particularly abundant in these two species.

 

[Sidisheikh.mehriz]

Addressing the variability in retardation presents an opportunity for enhancement. Exploring improved defatting methods holds promise in this regard.

However, a critical consideration arises: which solvents are optimal for selectively removing more polar constituents such as resins while preserving the integrity of alkaloid salts within the sample?

The utility of TLC is undeniable, offering rapid phenotyping at a super cost-efficient scale. Yet, for in-depth analyses of specific specimens, transitioning to HPLC appears prudent to unlock richer data insights.

Thus far, our analyses have predominantly focused on P. aquatica and P. arundinacea, yet we have not observed any 5-HO-DMT presence. This suggests that this compound may not be particularly abundant in these two species.

SPE (solid phase extraction) will be the answer. Specifically a strong cation exchange resin column. Can be bought as readily made cartridges or you can build the column yourself buying the resin beads.