Ashwagandha is a herb that belongs to the night shade family of plants. The nightshade family of plants is a very interesting one and includes a lot of vegetables that make up large parts of many diets. Notable nightshade members are potatoes, tomatoes, chili peppers, goji berries and even tobacco! Another name for the nightshade family is Solanaceae. This is where Ashwagandha gets its Latin name from; Withania somnifera. Ashwagandha is a short perennial shrub, with a large root system that develops small deep orange fruits. The fruits resemble a small cherry and the leaves look frosty due to the many tiny hairs that are on them. This one of the reasons why Ashwagandha is also referred to as ‘winter cherry’. Traditionally, the Ashwagandha root is used, however new research has found high concentrations of key components in the leaves too. Various manufacturers have taken notice of this. One of the most notable being Natreon who produce a specialized extract of Ashwagandha called Sensoril. Sensoril is made from the leaves of Ashwagandha, giving it a unique chemical composition, which produces noticeable calming effects.

If we advance a little bit more in the story of Bacopa monnieri, we start running into testing difficulties. As was mentioned earlier, the bacosides stick together due to their saponin nature, which makes it hard to separate them in the HPLC column. However, it was discovered that the bacosides could be acid hydrolyzed to ebelin lactone, which could then be easily quantified by UV-spectrophotometry. Then working backwards with some math, it could be determined how much bacoside was present. However, nobody stopped to think about how ebelin lactone was contributing to the effects of Bacopa monnieri at the time, and thus, this compound was briefly forgotten about again.

It does appear that the CDRI was aware of ebelin lactone and the other bacogenins, which are the hydrolyzed derivatives of bacosides. In fact, ebelin lactone is bacogenin A4! However, the research on the bacogenins remained sparse, and seemingly no solid attempts were made to concentrate the bacogenins. Then one day, we were diving deep into Bacopa monnieri research, and we came across a fascinating study from 2015. Within this study, it was determined that the bacosides likely did not absorb in the gut, nor did they have the ability to cross the blood brain barrier (BBB).

The fact that bacosides cannot absorb properly nor cross the BBB obviously poses some major problems. The main benefits of Bacopa monnieri happen in the brain, and thus the compounds responsible need to be absorbed and absolutely need to be able to cross the BBB! So, was everyone looking at the wrong compounds all along, or is there something else going on? Well, according to the 2015 study, the bacosides quite rapidly hydrolyze to their aglycone counterparts, jujubogenin and pseudojujubogenin. Jujubogenin and pseudojujubogenin do appear to absorb quite well in the gut, and also seem to cross the BBB. This initial deglycosylation step is quite easy, and thus it can likely be assumed that for Bacopa monnieri to be active, the bacosides must first be deglycosylated. However, if we now think back to what we mentioned about UV-spectrophotometry, where the bacosides were acid hydrolyzed to ebelin lactone to make analysis easier, it becomes clear that this process may also be happening in our stomach. The 2015 study verifies this to an extent, and more importantly, shows that ebelin lactone absorbs extremely well and has high BBB permeability!

The most interesting discovery in this 2015 study however, is that ebelin lactone acts at the 5-HT2A receptor. This is a major target within the practice of microdosing, a practice which has garnered A LOT of attention over the years. Highly influential researchers such as James Fadiman popularized the notion of microdosing, and before long it took the world by storm. However, it has some very obvious downsides associated with it for the average person, especially depending on where in the world you are located. Thus, it made us very excited that humble Bacopa monnieri could generate a compound that may perhaps have microdose-like effects! Another fascinating, but decidedly less sexy target that was discovered in the 2015 study, is that ebelin lactone acted at muscarinic acetylcholine M1 receptors. Within the research world on cognitive function, this is a bit of a holy grail target and it’s been very difficult to find a somewhat selective ligand for this receptor. So you can imagine just how excited we started to get about ebelin lactone! However, now came the hard part, sourcing it…

Now that we had our eye on ebelin lactone, we needed to figure out how to get our curious hands on this amazing compound! Well, this proved to be quite challenging. First, we looked around for a synthetic source, a strategy which we have employed often for “nature identical compounds' ' for example in our salidrosol solution where we use synthetic versions of salidroside and tyrosol. However, there was no synthetic ebelin lactone to be found and pricing wise, a custom synthesis was completely out of the question. Back to the drawing board it was!

That’s when it dawned on us that ebelin lactone was also being produced naturally in the human body, and that ebelin lactone likely plays a huge role in the overall cognitive effects of Bacopa monnieri. At the time of this initial research, Panax ginseng GS15-4 was also very popular, and the makers of this Panax ginseng extract did something incredibly unique. They mimicked the metabolism of the ginsenosides contained within Panax ginseng, via fermentation and yielded the more potent ginsenoside metabolites. This made us think, if GS15-4 was mimicking a process that is naturally occurring within the human body, then why can’t we?

We picked the metabolism process for the bacosides that is happening within the human body apart, and came up with a two step process. First, we hydrolyze the bacosides into their aglycone counterparts, jujubogenin and pseudojujubogenin. Then we apply a secondary acid hydrolysis step on the jujubogenin and pseudojujubogenin in order to yield ebelin lactone and the other bacognenins. A great idea, but easier said than done! Eventually we started working together with an Australian company that shared many of our values and had the capabilities to help us develop this two stage hydrolysis process. After many months, an initial pilot batch was made, packaged up and sent to us.

Once the pilot batch arrived, we immediately dosed 50 mg of the extract, and experienced effects that were somewhat similar to our Bacopa monnieir 24% bacosides extract, however the effects kicked in much quicker and there was an extra little sparkle there. Then we upped the dose to 100 mg and the effects got pretty interesting, however it was still similar to regular Bacopa monnieri and this is not exactly what we were after. After some analytical testing, we discovered that the second acid hydrolysis step wasn't complete, which meant that we now had a mix of jujubogenin, pseudojujubogenin and ebelin lactone. A new batch was started with a much more complete secondary acid hydrolysis stage, and we waited patiently.

A few months later, the second completely acid hydrolyzed extract was in our hands! Upon trying it, lots of beta-testers were not able to identify it as Bacopa monnieri. The sometimes overpowering calming effects of Bacopa monnieri were gone, and to those of us with microdosing experience, we felt stark similarities. We were on to something now! After extensive analytical testing we found that we had now achieved our goal, a fully acid hydrolyzed Bacopa monnieri extract, with effects that drastically set it apart from traditional Bacopa monnieri extracts. We were instantly focused, energized and just generally more present in the moment, this is exactly what we were aiming for!

Now came the hard part, patenting the extraction and processing method that yielded this ebelin lactone rich Bacopa monnieri extract. It already took almost two years to get to the final beta-test sample, and we knew the patent could take almost as long too. We hunkered down, sat down with a bunch of lawyers, all chipped in and in Novemeber 2022, we were finally able to submit the patent and release the extract, which we called Cognance! This was the story of Cognance, truly Bacopa reimagined!

*These statements have not been evaluated by the Food and Drug Administration. This product is not intended to diagnose, treat, cure, or prevent any disease.

Now that we’ve covered the story of cognance it is time to move onto the most exciting aspect of this novel extract, the effects and the science behind them! Strap in, because this is going to get very nerdy!

As we mentioned briefly in the story of cognance, the principle compounds within Bacopa monnieri, the bacosides, are not very bioavailable. Part of this is due to their sheer size! Take a look at the structure of bacoside A3 for example:

Visually, it’s huge! Molecularly, it is also quite heavy, clocking in at 929.10 g/mol. Big molecules tend to not absorb well which was made abundantly clear by Christopher A. Lipinski, a famous medicinal chemist. He stated that for a molecule to be absorbed, its molecular weight should be under 500 g/mol. This means that bacoside A3, is almost two times over Lipinski’s limit! This is where things get interesting though, because we can “shave” some weight off. On the left hand side of the molecule, there are three similar looking chemical structures. These are sugar groups, and are not particularly important to the effects of bacoside A3. If these sugar groups are removed, then we can remove a lot of molecular weight. This process is referred to as deglycosylation (removal of sugar groups) which then yields an aglycone (a compound with sugar groups removed). This means that we can refer to bacoside A3 as a glycoside (a compound with a sugar group), and when we remove the sugar groups we end up with the following compound:

This compound is called jujubogenin and can be considered to be an aglycone of bacoside A3. Now when re-evaluate the molecular weight of jujubogenin we find that 456.4 g/mol of sugar groups have been removed, which now means that jujubogenin clocks in at 472.7 g/mol, which is well below Lipinski’s limit of 500 g/mol! Indeed, we also see that the bioavailability now goes up considerably. Jujubogenin in contrast to bacoside A3, now shows good gastrointestinal absorption and blood brain barrier (BBB) penetration. This indicates that jujubogenin most certainly is very bioactive.

Interestingly enough, the bacosides deglycosylate in the human gut after human consumption, to jujubogenin as we just discussed, but also pseudojujubogenin. This means that after oral consumption of a Bacopa monnieri extract, you’d experience a significant delay in effects while the bacosides are being converted to their more active metabolites. We also had a sneaking suspicion that jujubogenin and pseudojujubogenin were responsible for the lethargy inducing effects of Bacopa monnieri. Part of this theory was based on research on jujubogenin indicating that it interacts with the GABA-A receptor. More specifically, two distinct subunits of the GABA-A receptor, alpha1 and alpha 5. Activation of both GABA subunit alpha 1 and alpha 5, can induce very strong relaxing effects. More interestingly we even discovered that jujubogenin could upregulate these GABA subunits over time, which corroborates with anecdotal reports that Bacopa monnieri can become progressively more lethargy inducing over time!

We got an opportunity to put this theory to the test with our first pilot batch of cognance. For this batch, a very complete deglycosylation reaction was carried out via hydrolysis. This yielded high concentrations of jujubogenin and pseudojujubogenin. However, the second acid hydrolysis step to turn jujubogenin into ebelin lactone (which normally also happens in the human gut) had not been very efficient in this first pilot batch and thus we had a more jujubogenin and pseudojujubogenin leaning extract. When we tried this extract out, we experienced very rapid and fairly strong relaxing effects which meshed well with the research we had read. After this experience, we knew we were on to something, and that if we were able to turn jujubogenin and pseujujubogenin into their bacogenin counterparts efficiently, then we could very well bypass the lethargy inducing effects!

This meant we had to better dial in the acid hydrolysis step. After some tweaking we finally got it to work! Our lab director drew up a fantastic graphic to show all of you chemistry nerds exactly what happened during this stage:

Once we got this second pilot batch in our hands, everything changed. The lethargy inducing effects were gone, and a new focus enhancing, creative energy replaced it. This is mostly due to ebelin lactone, so let’s discuss in detail exactly what it is doing!

Ebelin lactone is a very unique compound, with a pharmacodynamic profile that is not often found elsewhere. The most unique effect is that it acts as a positive allosteric modulator of the serotonin 5-HT2A receptor. What this means is that ebelin lactone binds to the allosteric site of the 5-HT2A receptor, which is distinctly different from the orthosteric binding site, which is where most serotonin 5-HT2A agonists or antagonists bind. When a compound binds to the orthosteric binding site of 5-HT2A receptors, it can either directly activate or deactivate the receptor. In contrast, when a compound binds to the allosteric binding site of the 5-HT2A receptor, it can either make the receptor more or less sensitive. With a positive allosteric modulator, like ebelin lactone, the sensitivity of the 5-HT2A receptor is increased. This means that it will require lower concentrations of ligands to activate the 5-HT2A receptor. The most relevant ligand in this scenario is the main endogenous ligand of the 5-HT2A receptor, serotonin. This is because serotonin will always be present, thus, enhancing the sensitivity of the 5-HT2A receptor with ebelin lactone will produce more serotonin induced activation of the 5-HT2A receptor.

There are numerous benefits to utilizing a positive allosteric modulator over a direct agonist. The first of which is that allosteric binding sites from receptor to receptor are often very different, while the orthosteric binding sites of receptors often have lots of similarities from receptor to receptor. This means that allosteric ligands oftentimes are much more receptor selective. The second advantage of a positive allosteric modulator, is that the effects can be less pronounced but more sustainable. In addition to this, there seems to be a ceiling to how much positive allosteric modulation can occur. In the case of the 5-HT2A receptor for daily functional use, this is very beneficial.

So, what does increased 5-HT2A activity provide? The 5-HT2A receptor is one of the most abundant serotonin receptors in the neocortex, especially in layer V of the cerebral cortex. These parts of the brain are absolutely crucial to cognitive function and sensory processing. Within these brain regions, 5-HT2A receptors have an excitatory effect, mostly through their effect on glutamate and dopamine release. With this in mind, it is clear to see why modulation of 5-HT2A receptors has become such a sought after concept. When we directly activate 5-HT2A receptors to a high degree, we see very extreme effects, especially on sensory processing and cognitive flexibility. While this can certainly be useful in certain situations, such high intensity of 5-HT2A receptor activation is undesirable for functional daily use. Due to this, alternatives were sought out, and the concept of microdosing was discovered, which basically takes high level 5-HT2A activation and minimizes the activation to the bare minimum. When this is done, the sensory processing and cognitive flexibility enhancement becomes incredibly useful!

This is where ebelin lactone is very unique, since it produces this exact low level increase in 5-HT2A activity, thereby closely mimicking microdose 5-HT2A effects. Thus, after dosing ebelin lactone via cognance, we can expect an uptick in sensory processing, coupled with increases in cognitive flexibility. This can help enhance focus, and divergent thinking, a concept many of us know as “creativity”. Furthermore, after periods of more extensive dosing, the increased sensitivity of 5-HT2A receptors may also result in memory enhancing activity.

Another very interesting aspect of ebelin lactone are its effects on the muscarinic acetylcholine M1 receptor. This receptor, similar to the 5-HT2A receptor, is absolutely crucial to cognitive function and memory. In fact, in many studies on cognitive function, a muscarinic M1 antagonist is often employed to simulate cognitive deficiencies. This muscarinic M1 antagonist is called scopolamine, and the study design is called a “scopolamine challenge”. When the muscarinic M1 receptor is blocked, major reductions in cognitive function and memory consolidation are observed. This is useful in study designs, because you can then try to overcome these reductions in cognitive function and memory consolidation with your target compound or plant extract. Ebelin lactone achieves the exact opposite effect however, and instead makes the muscarinic M1 receptor more sensitive rather than blocking it, once again acting as a positive allosteric modulator there.

One big issue with compounds that act on the muscarinic M1 receptor however, is that they are not very selective. This is problematic because the other muscarinic acetylcholine receptors can produce some very undesirable effects when activated, such as excessive sweating and salivation. It’s very hard to find selective muscarinic M1 agonists because the orthosteric binding sites between the different muscarinic acetylcholine receptors are all very similar. However, similar to the serotonin receptors, the allosteric sites on the muscarinic acetylcholine receptors are all fairly distinct from each other. Since ebelin lactone is acting as a positive allosteric modulator of the M1 receptor, this means that it can be fairly selective. Thus, providing powerful cognition enhancing effects without many if any downsides!

Similar to the 5-HT2A receptor, the muscarinic M1 receptor is also abundantly distributed throughout the neocortex, and also has an excitatory role here. While 5-HT2A has a more direct effect on sensory processing in these brain regions, muscarinic M1 has a more direct effect on memory consolidation. This means that when combined, the effects of positive allosteric modulation of both muscarinic M1 and serotonin 5-HT2A receptors, produces a very unique and comprehensive nootropic effect! We get the benefits of more cognitive flexibility and enhanced sensory processing, via 5-HT2A, while achieving higher levels of memory consolidation via muscarinic M1 receptors. Together this leads to a unique acute focus enhancing effect, and in the long run, this pharmacodynamic profile leads to enhanced cognitive performance and memory consolidation!