On December 10th, researchers from the D’Or Institute for Research and Education and two Brazilian universities published results from laboratory testing that showing that apigenin, a flavonoid found in many different plants, promotes healthy neuron formation and strengthens brain cell networks.

Apigenin

Apigenin

Researchers have been interested in the health benefits of apigenin for decades, but very little clinical testing has been conducted with humans. Previous animal testing and anecdotal evidence point to major potentials for the therapeutic use of apigenin.

 (AP Photo/Robert F. Bukaty)

(AP Photo/Robert F. Bukaty)

Apigenin is found in parsley, celery, chamomile, red pepper and many other plants, including the flowers of cannabis plants. Cannabis flowers contain many interesting terpenoid and flavonoid combinations, and recent scientific evidence suggests that whole plant extracts allow all of these compounds to act synergistically with one another.

Concentrated Apigenin

Concentrated Apigenin

In this article, we will look at a recent study that examines the specific flavonoid apigenin, which is also commonly found in cannabis. Smoke Reports wants to keep up the conversation about the medical potentials of all of the compounds within cannabis flowers, including cannabinoids, terpenoids, and flavonoids.

What is Apigenin?

Apigenin is a naturally-occurring plant compound of the flavone class. Apigenin has been shown to provide many pharmacological benefits, including autophagy in leukemia cells and apoptosis in the cells of rats with cyclosporin-induced renal damage.

“[Apigenin] has shown to decrease the secondary effects of ciclosporin A, an immunosuppressive administered during organ transplants to avoid the rejection of the transplanted organ. It has also been proven that apigenin is one of the few substances capable of stimulating the monoamine transporter, altering the neurotransmitter levels.

It has recently become clear that apigenin acts as an anxiolytic and sedative on the GABA receptors. The fact that this effect is shared by the cannabinoids bring us to a possible synergy between anxiolytic and sedative effects of cannabinoids.”

– Fundación Canna

Apigenin is unlike other flavonoids in that is acts as a monoamine transporter (MAT) activator, making it one of only a handful of chemicals that are responsible for the re-uptake of dopamine, norepinephrine, and serotonin. MATs are thought to play a significant role with conditions like ADHD, depression, Parkinson’s disease, Schizophrenia, and Tourette’s syndrome.

Apigenin and Improved Neuron Functioning

In the recent study out of Brazil, (Commitment of Human Pluripotent Stem Cells to a Neural Lineage is Induced by the Pro-Estrogenic Flavonoid Apigenin) researchers examined the positive effects of apigenin when applied to human stem cells.

In a matter of days, the stem cells that had been exposed to apigenin had turned into neurons, and these neurons were capable of stronger and more robust connections with other brain cells.

the brain

Previous animal studies involving apigenin show that flavonoids are capable of improving memory and learning functions, yet this Brazilian study is the first to apply apigenin directly to human cells. In this particular case, the researchers demonstrated the ways in which apigenin works within the human body:

“In particular, apigenin (API) has been shown to bind to estrogen receptors, which affect the development, maturation, function, and plasticity of the nervous system. The aim of this study was to investigate the effects of apigenin (4′,5,7-trihydroxyflavone) upon the neural differentiation of human pluripotent stem cells.”

-Abstract of the study: Commitment of Human Pluripotent Stem Cells to a Neural Lineage is Induced by the Pro-Estrogenic Flavonoid Apigenin

The study was conducted as part of the PhD dissertation of Cliede Souza of the Institute of Biomedical Science, at the Federal University of Rio de Janeiro, Brazil. Other participating researchers include Bruna Paulsen and Helena Borges, both of the Federal University of Rio de Janeiro, as well as Silvia Costa of the Federal University of Bahia. Stevens Rehen and Sylvie Devalle both contributed to the study from the D’Or Institute of Research and Education.

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Images from the Apigenin Study

The following images can be viewed in their entirety at the original source, along with the full text of the study.

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Fig. 1. Apigenin (API) enhances the expression of human neural precursor cell markers. Representative immunofluorescence images for detection of neural precursor cell markers nestin (NES; green, a and c) and SOX2 (green, b and d) counterstained with DAPI (blue). Human embryonic stem (hES) cells treated with 10 µM API for the six initial days of culture showed intense immunostaining for (c) NES and (d) SOX2. Bar graph showing the percentage of NES (e) and SOX2 (f) positive cells in each treatment group. Control cells (cells cultured without API) displayed few cells staining for both markers: (a) NES (p=0.00103) and (b) SOX2 (p=0.00421). Scale bar=50 µm. The values are expressed as the mean±SEM; n=3. (source).

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Fig. 2.   Estrogen receptor (ER) activation is required for API-induced differentiation into neural progenitors. Representative immunofluorescence images staining for (a) NES (green) counterstained with DAPI (blue). hES cells were treated with 10 µM API for the six initial days of culture in the presence of ER antagonists applied starting 2 h prior to API treatment. The antagonists used were (b) 10 nM methyl-piperidino-pyrazole (MPP; ESR1) and (c) 1 µM pyrazolo[1,5-a]pyrimidine (PHTPP; ESR2). API-induced neural differentiation was inhibited by ER antagonists. A reduction in the number of NES+ cells was observed after treatment with MPP (one-way ANOVA, p=0.0000833769) and with PHTPP (one-way ANOVA, p=0.00478). (d) Bar graph showing the percentage of NES+ cells in each treatment group; the values are expressed as the mean±SEM; n=3. Scale bar=50 µm. (source).

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Fig. 3.   API modulates retinoic acid receptor (RAR) expression. Images of dual labeling for NES (green) and RARA (red) show more intense staining for both proteins in hES cells (b) treated with 10 µM API compared to (a) untreated control cells. (c) The fluorescence intensity as a percentage of the total number of cells shows significantly increased RARA staining in API-treated cells compared to untreated controls (p=0.00047). (d) Western blotting showed increased expression of RARB (p=0.00276) and RXRG (p=0.00633), but not for RARG1 (p=0.10777), RXRA (p=0.70516), or RXRB in treated cells. Quantification of Western blotting for (e) RARB, (f) RARG1, (g) RXRA, and (h) RXRG are shown. The results were normalized relative to a control group considered as 100% and at least three independent experiments. The values are expressed as the mean±SEM; n=3, t-test. Scale bar=50 µm. (source).

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Fig. 4.   RARs participate in neural differentiation. hES cells were treated with 10 µM API for the first 6 days of culture (18 days total) (a). RAR antagonists were applied to cell cultures starting 2 h before and during the entirety of API treatment. Images show immunofluorescence staining for NES (green) counterstained with DAPI (blue). API-induced neural differentiation was inhibited by RAR antagonists. A reduction in the number of NES+ cells was observed after treatment with (b) 10 µM ER 50891 (p=0.00701), (c) 10 µM LE 135 (p=0.000281), (d) 10 µM MM 11253 (p=0.001171), and (e) 10 µM UVI 3003 (p=0.001787) compared to (a) cells treated with API alone. Quantification is shown in (f). The values are expressed as the mean±SEM; n=3 (one-way ANOVA). Scale bar=50 µm. (source).

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Fig. 5.   API induces human neuronal differentiation. Neural progenitors obtained from hES cells treated with API were allowed to differentiate for an additional 25 days in medium without API (see Methods in the Supplementary file). Undifferentiated hES cells were used as a negative control. Phase-contrast microscopy (a, b) or immunofluorescence staining for the following neuronal markers: (c, d) microtubule-associated protein 2 (MAP2), (e, f) synapsin 1 (SYN1), (g, h) calretinin (CALB2), (i, j) β-tubulin class III (TUBB3), (k, l) polysialic acid form of neural cell adhesion molecule (PSA-NCAM), (m, n) neurofilament (NEF), and (m, n) myelin basic protein (MBP), (o, p) synaptophysin (SYP), and (o, p) DLG4. Scale bar=50 µm. (source).

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Fig. 6.   Distinct neuronal and glial markers after API neuronal differentiation. Neuronal diversity induced by API treatment is characterized by the presence of specific neuronal subtype markers such as (a, b) choline acetyltransferase (CHAT), (a, b) glutamate decarboxylase (GAD1), (c, d) glial fibrillary acidic protein (GFAP), (c, d) brain lipid-binding protein (FABP7), (e, f) parvalbumin (PVALB), and (e, f) tau protein (MAPT). Pluripotent hES H9 cells were used as a control and were negative for all neuronal and glial markers (a, c, e). Scale bar=50 µm. (source).

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Fig. 7.   API augments formation of synapses. Confocal microscopy of dual staining for the synaptic markers SYP (red) and DLG4 (green). Retinoic acid-induced neurons were treated with 1 µM API for 72 h. Untreated (A and A′) and API-treated (B and B′) cells presented neuronal morphology and were positively stained for SYP and DLG4. (b) API-treated cells showed more intense staining for both markers compared to (a) the untreated group. Insets show high-magnification images of selected areas in A and B with co-localized puncta in yellow (A′ and B′). (c) Higher co-localization indexes were measured after API treatment (t-test, p=0.0016; n=3). (d–f) Levels of synaptic protein expression were evaluated by Western blotting. Western blotting showed increased expression of SYP (p=0.0063287) but not DLG4 (p=0.287319) in treated cells. The results were normalized relative to a control group considered as 100% and at least three independent experiments. The values are expressed as the mean±SEM. Scale bar=50 µm. (source).

Apigenin and Other Flavonoids: Full Spectrum Cannabis Synergy

Apigenin is found in a number of plants, and this particular study ultimately recommends further research into the health benefits of a flavonoid-rich diet. Yet food is not the only way to absorb flavonoids. In fact, cannabis flowers contain a particularly large quantity of flavonoids, with experts at the Fundación Canna estimating that up to 2.5% of the dry flower weight could be flavonoid content.

Resinous Glands on the Cannabis Flower Produce Cannabinoids, Terpenoids, and Flavonoids like Apigenin

Resinous Glands on the Cannabis Flower Produce Cannabinoids, Terpenoids, and Flavonoids like Apigenin

It is strongly suspected that cannabinoids, terpenes, and flavonoids all work together to provide synergistic health benefits. Clinical testing has shown that the effectiveness of an individual compound will plateau, whereas the combination of these plant compounds continues to provide health benefits as the dose increases.

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The distribution and concentration of flavonoids in cannabis has been largely ignored by the medical community, but the mounting evidence shows that cannabis patients should consider flavonoids as an important factor in their cannabis therapy.

Bringing Apigenin to the Cannabis Conversation

Cannabis science is improving, slowly but surely. Several thousands of years worth of medical acumen was literally wiped away during the last eight decades of cannabis prohibition, yet researchers and doctors are both actively investigating the potential health benefits of the many compounds found in cannabis.

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Cannabinoids like THC (tetrahydrocannabinol) and CBD (cannabidiol) are still viewed as the primary components of a federally-illegal narcotic, creating many obstacles for researchers pursuing substantial knowledge. However, compounds like flavonoids and terpenoids are found throughout many plant organisms, and therefore are not automatically associated with psychoactive cannabis.

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Common Fears and Misconceptions about Reactions to Cannabis

Cannabis medicine is so exciting because even the lack of clinical evidence has not kept positive medical results from making themselves clear. When studies are published that are not specific to cannabis, but involve positive medical benefits associated with cannabis flowers, it is important that our community recognize and embrace the full spectrum of cannabis plant constituents.

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Smoke Reports supports all cannabis studies, because clinical data will help all of us to better understand our relationship with cannabis. Check out our blog for future examinations of cannabis research.

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