Sunday, September 2, 2012

Future Pharmacy: Amentoflavone

Amentoflavone is a polyphenolic compound extracted from many different plants including Ginkgo biloba and St John's Wort. It has received most of its attention due to its anti-cancer and anti-microbial properties. This article will focus on its lesser known mechanisms which are more relevant in the context of athletic performance.

I first became aware of amentoflavone in 2005 when a research article was published demonstrating its ability to negatively modulate the benzodiazepine GABA(A) receptor site (1). This modality is a potential way to enhance learning and memory since it would disinhibit excitatory neurotransmission. It could also increase the production of testosterone by inducing the release of GnRH at the hypothalamus. Unfortunately, as its structure should remind us, amentoflavone has almost no capacity to cross the blood brain barrier as was demonstrated in the 2008 study by Colovic et al (2). This is not necessarily terrible news, as GABA(A) antagonism brings along with it a host of potentially dreadful side effects from anxiety to the potential for neurotoxicity. Luckily, its peripheral properties are significant enough to warrant further investigation.

Phosphodiesterase Inhibition
Phosphodiesterase (PDE) is an intracellular enzyme which degrades the second messengers cAMP or cGMP. In human adipose tissue, beta-2 agonism results in an increase in cAMP which activates lipases that cause cellular fat breakdown ("lipolysis"). By inhibiting the particular phosphodiesterase isoenzyme (PDE3) found in adipose tissue, a compound could theoretically synergize with the adrenergic signaling cascade and induce significant fat loss. Indeed, amentoflavone has demonstrated this capacity in a 1998 Italian study examining the effect of Ginkgo biloba on rat adipose tissue (3).
This work compares the inhibition of cAMP-phosphodiesterase in rat adipose tissue by a mixture of Ginkgo biloba biflavones with the effect of individual dimeric flavonoids. The degree of enzyme inhibition by G. biloba biflavones was amentoflavone > bilobetin > sequoiaflavone > ginkgetin = isoginkgetin. 
A 2006 Planta Medica article also identified amentoflavone as a weak inhibitor of PDE5, although having much greater inhibitory capacity for other isoforms (4, 5). The former PDE is responsible for the metabolism of cGMP, whereas the latter isoforms deal mainly with cAMP. Inhibiting cGMP disposal allows for vascular dilation (i.e. Viagra) via smooth muscle relaxation. Inhibiting cAMP metabolism potentiates various transduction cascades including lipolysis in adipose tissue, as discussed above, and enhancing cardiac contractility and speed (6).

Muscular Strength
Amentoflavone was recently demonstrated to possess acetylcholinestase inhibiting properties in a 2011 study (7). By inhibiting AchE, more acetylcholine ligand would be available at the neuromuscular junction, disinhibiting Ach metabolism from being a rate limiting step for muscular contraction. Unfortunately, AchE inhibition alone has not demonstrated an ability to enhance muscular strength in healthy individuals (8). Fortunately, however, amentoflavone possesses another modality that may synergize well with AchE inhibition: enhancing calcium release from the sarcoplasmic reticulum.
The Ca2+ -releasing activity of amentoflavone was approximately 20 times more potent than that of caffeine...These results suggest that amentoflavone, which does not contain a nitrogen atom, probably binds to the caffeine-binding site in Ca2+ channels and thus potentiates Ca2+ -induced Ca2+ release from the heavy fraction of fragmented sarcoplasmic reticulum. 
This is a novel mechanism for enhancing muscular contraction and one of the ways in which caffeine increases strength, albeit weakly (9). Since amentoflavone is approximately 20 times more potent then caffeine, it is also possible that it could exert greater efficacy in this area.

Other Mechanisms
Amentoflavone, in addition to its exceptionally weak ability to inhibit fatty acid synthase (10) and ability to potentiate cAMP in adipose tissue, also possesses another novel metabolic mechanism: Protein tyrosine phosphatase 1B (PTP1B) inhibition (11).

Regulation of protein phosphatases in disease and behaviour; EMBO reports (2003) 4, 1027 - 1031 doi:10.1038/sj.embor.7400009
PTP1B is an negative regulator of the growth promoting cascade induced by tyrosine kinase receptors. By inhibiting PTP1B, amentoflavone disregulates the downstream pathways activated by various ligands, including those induced by insulin. This could have an exceptionally beneficial effect in relation to insulin insensitivity, or just as a means to potentiate insulin itself. Unfortunately, it could also have pro-oncogenic outcomes in those with cancer. Needless to say, any growth promoting compound (estrogen, GH, IGF-1, DHT, et cetra) has the capacity to stimulate oncogenesis, and so this mechanism should not be hysteria-provoking - especially in light of amentoflavones other anti-cancer modalities (anti-mutagenesis, anti-angiogenesis).

  • PDE inhibition (multiple isoforms)
    • Weakly vasodilatory
    • Capacity to potentiate adrenergic signaling in adipose tissue --> enhanced lipolysis
  • Acetylcholinesterase inhibition
    • Increased availability of acetylcholine at the NMJ
  • Enhancing the release of Ca2+ from the sarcoplasmic reticulum
    • Increased contractility of skeletal muscle
  • Inhibition of PTP1B
    • Potentiation of insulin signaling and other growth promoting cascades (unknown tissue specificity)



  1. Pulsed ultrasound may be a way to make the BBB more permeable to substances.

  2. Wnt/β-catenin signaling controls development of the blood–brain barrier

  3. is there a possibility of contacting you by email? I'm a budding young Irish chemist that has a massive interest in your work. I've been reading it for over a year now and I was curious as to what your background is and how ya got into doing what you are doing?

  4. For being a, "pharmacology", blog, it seems a very fundamental and elementary principle in pharmacology is neglected. That is, the concentration utilized in these in vitro studies must be taken into account if attempting to extrapolate what may happen in vivo.

    Contrary to what is written in this blog, caffeine DOES NOT enhance muscle strength by increasing calcium release from the SR, as the concentrations shown in vitro to do so require MILLIMOLAR concentrations to elicit such an effect (1). A human would have to ingest supraphysiologic and obviously toxic/deadly quantities of caffeine to achieve such concentrations (i.e., 25-30 grams).

    So, sure amentoflavone may in fact being 20 times more potent than caffeine with respect to enhancing calcium release in vitro, but when you consider that the concentration for caffeine shown to increase calcium release requires amounts to be ingested that no human could survive without hospitalization, what is relevancy?

    So, even blindly assuming that it has equivalent bioavailabiilty (assuming any pk data exist) as that of caffeine, amentoflavone would only require 1,500 mg or more to equal such an effect. And what sort of toxicity data are available at a dose such as this? And what company sells this compound at such a dose?

    The lesson to be learned for people that may not know better is that simply because a paper is published showing a given pharmacological effect at a particular concentration, DOES NOT mean it is achievable in vivo.


    "The Ca2+ -releasing activity of amentoflavone was approximately 20 times more potent than that of caffeine...These results suggest that amentoflavone, which does not contain a nitrogen atom, probably binds to the caffeine-binding site in Ca2+ channels and thus potentiates Ca2+ -induced Ca2+ release from the heavy fraction of fragmented sarcoplasmic reticulum.

    This is a novel mechanism for enhancing muscular contraction and one of the ways in which caffeine increases strength, albeit weakly (9). Since amentoflavone is approximately 20 times more potent then caffeine, it is also possible that it could exert greater efficacy in this area."

  5. What evidence do you have that caffeine ->REQUIRES<- MILLImolar concentrations in order to increase intracellular calcium? The study you posted ->ONLY TESTED<- 2 caffeine concentrations. Perhaps you need to brush-up on your literature review?

    Here is an EN VIVO -> HUMAN <- study:

    "The tensions developed with electrical stimulation at lower frequencies increased significantly with caffeine ingestion, shifting the frequency-force curve to the left, both before and after fatigue. Mean plasma caffeine concentration associated with these responses was 12.2 +/- 4.9 mg/l. We conclude that caffeine has a direct effect on skeletal muscle contractile properties both before and after fatigue as demonstrated by electrical stimulation."

    And another en vivo human study:
    "Caffeine potentiated the force of contraction during the final minute of the 20-Hz stimulation (P < 0.05) with no effect of habituation. There was no effect of caffeine on 40-Hz stimulation strength nor was there an effect on maximal voluntary contraction or peak twitch torque. These data support the hypothesis that some of the ergogenic effect of caffeine in endurance exercise performance occurs directly at the skeletal muscle level."

    Physiological basis for caffeines enhancement of muscular strength in humans (2011 study):

    The authors commented:

    The action of caffeine on the calcium ion release in
    skeletal muscle may be responsible for peripheral
    manifestation of caffenism such as tremulousness, muscle
    twitching and hyperreflexia and may be managed with
    ryanodine receptor antagonist Dantrolene, Ruthenium,
    Procaine and Tetracaine.

    Since amentoflavone is more potent then caffeine in this regard (at least according to 2 studies), then I think it is worth mentioning. Its pharmacokinetic parameters don't have to be fully elucidated to speculate about a chemicals potential pharmacodynamics.

  6. What evidence do you have that caffeine is able to increase calcium release from the SR in skeletal muscle at physiologic concentrations?

    I have in fact reviewed the literature quite thoroughly and the lowest concentration I’ve seen is 5 mM, which is far greater than any concentration a human could achieve.

    You may want to re-read my comment again. I did not say that caffeine isn’t able to increase muscle contraction or muscular force. I said that it isn’t able to do so by affecting calcium release.

    Your premise was that since caffeine increases muscular force via increased calcium release, and amentoflavone is 20 times more potent at increasing calcium release, then amentoflavone must produce greater efficacy than caffeine in terms of muscular force. I am simply pointing out that there is no evidence that amentoflavone could affect muscular force because caffeine does not affect calcium release at concentrations that any human could achieve without being hospitalized. Thus, if calcium release is not the mechanism behind the increased force seen with caffeine, there is no reason to believe that amentoflavone would have any such benefit, let alone be more efficacious.

    I would strongly suggest that you read the following paper as it shows quite clearly that caffeine does not affect calcium handling at physiologic concentrations. It also serves as a rather good mini-review paper.

    What is with the author quotes? It’s almost as if you use them as though citing actual data in a paper or something. I can tell you first hand that reviewers don’t mind if you speculate or offer opinion, so long as it is clear. It’s generally denoted with the use of words like, “may”, “potentially”, or “possibly”. In fact, in your quote, this is evident. “The action of caffeine on the calcium ion release in skeletal muscle MAY be responsible for peripheral manifestation of caffenism….”. Yet, quite interestingly, this paper you cite and quote actually supports my position (see final note below).

    Regarding pharmacokinetic variables, they most certainly do matter if you’re going to discuss the potency of the two compounds and then argue that the amentoflavone may have greater efficacy than the caffeine. If amentoflavone is 20 times more potent than caffeine, but possesses only 5% of the bioavailability of caffeine, it isn’t so superior after all is it?
    Again though, the pharmacokinetics in this case don’t matter as there is good evidence that caffeine does not affect calcium release from the SR of skeletal muscle at physiologic concentrations, hence there is no reason to believe amentoflavone would yield any sort of benefit like caffeine.
    My own opinion is that caffeine’s ergogenic effects are likely due to adenosine antagonism or catecholamine release, though the latter likely declines with continued use.

  7. And to stick with your preference to post links to abstracts and quote authors, below is a link to a 2009 review paper in which the authors bring up the exact point I’ve been trying to get across here (read the first two sentences below):

    “Other mechanisms for caffeine have been suggested, such as enhanced calcium mobilization and phosphodiesterase inhibition. However, a normal physiological dose of caffeine in vivo does not indicate this mechanism plays a large role. Additionally, enhanced Na+/K+ pump activity has been proposed to potentially enhance excitation contraction coupling with caffeine. A more favourable hypothesis seems to be that caffeine stimulates the CNS. Caffeine acts antagonistically on adenosine receptors, thereby inhibiting the negative effects adenosine induces on neurotransmission, arousal and pain perception. The hypoalgesic effects of caffeine have resulted in dampened pain perception and blunted perceived exertion during exercise. This could potentially have favourable effects on negating decreased firing rates of motor units and possibly produce a more sustainable and forceful muscle contraction. The exact mechanisms behind caffeine's action remain to be elucidated”

    This paper would be good to read as well. The same conclusion is reached. There are in fact many papers out there that have reached the same conclusion. I feel odd having to point this out. This notion of calcium handling playing a role in caffeine’s effects in humans has largely been discarded by most authors, similar to the notion that was largely discarded about two decades ago regarding caffeine being able to inhibit phosphodiesterase as a potential mechanism. Again, the concentrations required in vitro to do so are far beyond what humans achieve.

    Last, I must point out that the only paper you reference (Olorunshola and Achi, 2011) which discusses the notion of calcium release being responsible for caffeine’s effect upon muscle force actually supports my position and the position of every published author that has addressed the issue. The paper you cite found that at least 15 mg/mL was required. This is approximately 1,000 times greater than the peak concentration seen after a 500 mg dose in humans! That would require around 500 grams. I sure hope no human would ever ingest that amount!

  8. "I have in fact reviewed the literature quite thoroughly and the lowest concentration I’ve seen is 5 mM, which is far greater than any concentration a human could achieve."

    One of the reasons why researchers use such high amounts is to increase the signal-to-noise ratio. Most of the time they are not trying to replicate physiologic doses. Secondly, the idea that caffeine's ability to enhance muscular contraction through central adenosine antagonism is less reasonable since caffeine's effects have been isolated to the periphery (see my second en vivo study), and that depleting intracellular caffeine negates its effects (less important). Thirdly, its ability to increase catecholamines mostly the same mechanism (i.e. calcium release) that is responsible for its direct skeletal muscle effects.

    Increasing >potency< means that at compound can equal maximal effect of another drug at a lower dose. (This effect can counterbalance a diminished bioavailability.) So, since at least 1 study has shown that caffeine's ability to induce SR calcium release requires ~250 micromol, and that a dose of 250 mg caffeine equates to roughly 100 micromol/L, since amentoflavone (all other PK values being equal)is 20x more potent, then it could require a much lower dose to cause the same effect. Furthermore, if it had a larger volume of distribution (albeit unlikely), then it could obtain a higher compartment concentration.