Description

7,8-Dihydroxyflavone Hydrate (DHT) Nootropic Liquid

 

 

 

 

CAS Number	38183-03-8
Other Names	7,8-dihydroxyflavone, 38183-03-8, 7,8-dihydroxy-2-phenyl-4H-chromen-4-one, 7,8-DHF, EINECS 253-812-4, ADB6MA8ZV2, BRN 0234350, CHEMBL75267, NSC-750341
IUPAC Name	7,8-dihydroxy-2-phenylchromen-4-one
Molecular Formula	C₁₅H₁₀O₄
Molecular Weight	254.24
Purity	≥99% Pure (LC-MS)
Liquid Availability	30mL liquid (10mg/mL, 300mg bottle)
Powder Availability	1 gram
Gel Availability	N/A
Storage	Store in cool dry environment, away from direct sunlight.
Terms	All products are for laboratory developmental research USE ONLY. Products are not for human consumption.

 

What is 7,8-Dihydroxyflavone Hydrate?

7,8-dihydroxyflavone hydrate is a nootropic compound categorized as a selective TrkB receptor agonist. The molecular makeup of the compound includes a catechol group that is crucial for enhancing the agonistic activity. Activation of TrkB is important considering that it binds to brain-derived neurotrophic factor (BDNF) in order to trigger dimerization and autophosphorylation of tyrosine residues. This ultimately results in activation of MAPK, PI3K and PLC-gamma-1 signaling pathways that are associated with the effectiveness of antidepressant drugs [1].

 

Main Research Findings

1) Treatment with 7,8-dihydroxyflavone hydrate is capable of improving spatial memory and increasing spine density in an animal model of neuronal loss similar to Alzheimer’s Disease.

2) By reducing retinal cell death, treatment with 7,8-dihydroxyflavone hydrate has the potential to improve visual functioning in a vertebrate model of inherited vision loss.

 

Selected Data

1) The research team of Castello et al analyzed GFP-labeled neurons in mice in order to assess how treatment with 7,8-dihydroxyflavone hydrate improves cognitive functioning in animal models of Alzheimer’s Disease. Female, homozygous Tet-DTa mice were crossed with homozygous Thy1-GFP-M male mice in order to produce a GFP-expressing CaM/Tet-DTa mouse. Once the desired homozygosity was reached the mice were maintained on a diet of 2000 parts per gram doxycycline to suppress induction of DTa. DTa expression was then induced by replacing 2000 ppg doxycycline with normal mouse chow for 25 days. To override DTa expression the chow was replaced and the mice were given 2 mg/ml doxycycline-supplemented drinking water for 2 days. After 2 days, the mice continued eating doxycycline chow but were given regular drinking water. Following the initiation of experimental protocol, the mice were subjected to several behavioral procedures in order for the researchers to examine changes in cognitive functioning [2].

At the beginning of the behavioral test all mice, 3.5-5 months-old were age and sex matched from 4 different independent litters in order to avoid litter effects. The elevated plus maze test was conducted first in order to assess anxious behavior in the animals. The maze apparatus was elevated 40 cm above the ground and consisted of 4 intersection arms, 2 of which were closed with dividing walls and 2 that were open with no dividing walls [2]. The animals were placed into the apparatus at the junction of the arms and allowed to explore the maze for 5 minutes while being recorded in order for the researchers to determine the total number or arm entries and how much time was spent inside of the arms.

Next, to assess for motor deficits the animals were subjected to rotarod testing. The animals were tested on a rotarod apparatus that accelerated linearly from 4 to 40 rpm over 5 minutes. Once the animal fell off of the rotarod or were able to hold on to the apparatus for 2 revolutions without walking, the trial was considered over. Each animal underwent 3, 5 minute trials each separated by a 10 minutes resting period. The elevated plus maze test and the rotarod testing was followed by water maze testing that included a 1 meter diameter circular pool filled with opaque water. During the training period the mice were placed in the pool and allowed to find and climb onto a submerged platform. After the 4 day training period ended the escape platform was removed and the mice were tested on a recorded probe trial 24 hours later [2].

After behavioral testing was concluded the mice were anesthetized using sodium pentobarbital and transcardially perfused with PBS at a rate of 11 ml/minute. The brains of the animals were then dissected and fixed in 4% paraformaldehyde for 48 hours and cryoprotected in 30% sucrose. A subset of the collected tissues were randomly selected for further histological analysis. Free floating sections were washed and incubated in PBS for 1 hour, followed by overnight incubation in primary antibodies for MAP2, NeuN, or synaptophysin. After the overnight incubation Alexa Fluor 555 fluorescent secondary antibody was applied for an additional hour of incubation time. The sections were finally mounted on slides for confocal microscopic imaging [2].

2) The research team of Daly et al examined the ability of the nootropic compound, 7,8-dihydroxyflavone hydrate to treat impaired visual functioning. For the purpose of the study male zebrafish with an AB genetic background were utilized. The subjects were treated 4 times per day for 1 hour with 3 mM ENU diluted in 10 mM sodium phosphate buffer in 7-14 day intervals. The treated male zebrafish were mated and the resulting embryos were used to generate wildtype fish that were screened for recessive mutations. Any fish carrying the recessive mutations were bred with wild type Tubingen fish in order to produce hybrid carriers that were then incrossed with mutant and normal offspring for further phenotyping and genotyping [3].

The heterozygous dye larvae were separated from other sibling larvae based on their phenotypes. All mutants and siblings were treated with 1 uM Trichostatin A 10 uM MCl1568, 10 UM MS275, 10 uM 7,8-dihydroxyflavone hydrate or a DMSO vehicle control. For each experimental treatment group, 12 larvae were transferred to 10 mL of the drug solution in a petri dish that was sealed and incubated under standard conditions until reaching 5 days post-fertilization. Once the samples reached 5 days post-fertilization, the HDACi-treated larvae were removed from the drug solutions and placed in an embryo medium for behavioral analyses [3].

The first analysis that was performed was the optokinetic response assay that involved transferring individual larvae to a petri dish containing 9% methylcellulose to immobilize the subjects. The dish was then placed into a circular grated pattern composed of black and white stripes. The pattern was then rotated clockwise for 30 seconds and counter-clockwise for an additional 30 seconds at 18 rpm while the number of saccadic eye movements per minute was recorded by the research team. The next analysis performed was the visual motor response assay where the treated larvae were transferred to an individual well containing 600 uL embryo medium [3]. The wells were placed in a recording chamber including a motion detection infrared camera that allowed for the researchers to quantify locomotor activity in response to changing light conditions.

The subjects underwent electroretinography next. The larvae were allowed 30 minutes to adapt to a dark environment prior to being paralyzed with 0.5 mg/mL of mivacurium chloride. The recording electrode was filled with a 0.9% saline solution and positioned onto the center of the cornea while the reference electrode was placed in E3 medium.
The dark-adapted status of the subjects was maintained throughout the handling and measurement process of the experiment and a 300 w halogen light source was used to provide light stimulation. Three optical density filters were used to produce flash intensities at -3.0, -2.0, -1.0, and -0log with a flash duration of 20 ms, followed by the completion of appropriate analysis and comparison of the raw data collected [3].

After the optokinetic response assay, visual motor response assay, and electroretinography were complete, 4 and 5 days post-fertilization HDACi-treated or control larvae were euthanized and transferred to a protease inhibitor solution. The eyes of the subjects were then enucleated and sonicated at 5% amplitude for 10 to 15 seconds in an 80 uL solution of protease inhibitor solution or lysis buffer. 20 ug of protein extracts were prepared, boiled, and centrifuged for 8 minutes to remove any excess pigment or cell debris. The protein samples were then separated and transferred to a fluoride membrane for 1 hour in a transfer buffer composed of 25 mM Tris base and 0.2 M glycine [3].

After an hour the membranes were blocked for 1 hour in a 5% milk solution and probed with several primary antibodies, including: acetyl-Histone, BDNF, AkT, and beta-actin. Following probing with antibodies, the membranes were washed in PBS-T and incubated in horseradish peroxidase conjugated secondary antibody for 2-4 hours. PBS-T washes were repeated and the membranes were placed in chemiluminescence western blotting detection reagent in order for the researchers to record activity of horseradish peroxidase using a bioluminescence imager. Densitometry was performed and relative optical-density was normalized to total Akt or beta-actin expression [3].

 

Discussion

1) The research team of Liu et al investigated the ability of 7,8-dihydroxyflavone hydrate to improve spatial memory and anxiety-like behavior in animal models. 5 mg/kg of the nootropic or a vehicle was intraperitoneally injected to CaM/Tet-DTa mice and Tet-DTa mice over the course of 2 weeks. During the 2nd week of treatment the animals were tested using an elevated plus maze. The researchers found that control mice spent 17% of the 5 minute trial in the open arms while the mice with induced synaptic loss were found to spend 61% of the 5 minute trial in the open arms of the maze. However, the groups spent an equal amount of time exploring the maze apparatus [2].

Figure 1: Changes in A) the amount of time spent and B) the number of entries in the open arms of the elevated plus maze apparatus by control and lesioned animals treated with a vehicle of 5 mg/kg of 7,8-dihydroxyflavone.

Overall, it was found that treatment with 7,8-dihydroxyflavone did not result in a significant change in spatial memory measured by the elevated plus maze test. Additionally, to determine if the DTa-induced lesion affects motor coordination the animals underwent an accelerated 3-trial protocol of rotarod testing. The lesion was not found to cause motor deficits as all groups were able to stay on the rotarod for equal amounts of time.

Figure 2: Changes in the time it took for the animals to fall off of the rotarod testing apparatus over 3 trials.

In addition to the elevated plus maze test and the rotarod testing, the animals underwent a water maze test in order for the research team to observe the impact of TrkB agonism on behavior dependent on hippocampal activity. The results of the water maze found that the DTa-induced lesioned mice treated with a vehicle were slowed to find the hidden platform on days 2-4 of the training period. Comparatively, by day 4 the lesioned mice treated with 7,8-dihydroxyflavone hydrate found the hidden platform significantly faster than the control group. As for the escape latencies of the mice, on days 2 and 4 there were no significant differences between the lesioned mice treated with a control versus those treated with the nootropic [2].

Figure 3: Changes in the amount of time it took to reach the hidden escape platform during the water maze test over 4 days of experimental trials.

24 hours after the final training day the mice were assessed on a 1-minute probe trial where the researchers removed the hidden platform in the water maze. The lesioned mice were slower than the control animals to reach the former location of the platform, and were typically much further from the previous location. That being said, probe latency for the lesion mice treated with 7,8-dihydroxyflavone hydrate was similar to control animals and significantly lower than the lesioned mice treated with a vehicle. The lesioned mice treated with the nootropic were also typically closer in proximity to the prior location of the platform in comparison to the lesioned mice treated with a vehicle. It was also noted by the researchers that video tracking software was used to record the swimming activity of the animals to ensure there were no differences in motivation or swimming ability that could potentially skew data [2].

Figure 4: Changes in E) latency, F) mean proximity, and G) swim speed of the control and lesioned animals treated with either a vehicle or 7,8-dihydroxyflavone hydrate, during the 24 hour probe trial.

As it was previously mentioned, the mice were euthanized after the behavioral testing was complete in order for the research team to measure the extent of neuronal loss in the lesioned animals by conducting immunohistochemistry for the neuronal nuclei marker, NeuN. In lesioned mice expressing GFP, the animals were shown to have 71% less immunoreactivity for NeuN in the CA1 pyramidal layer. These results were similar to those found in lesioned mice that did not express GFP that experienced a 74% reduction in CA1 pyramidal neurons. These findings indicate that introducing the Thyl-GFP transgene does not alter the functioning of the inducible lesion systems. The researchers also mentioned that there was a significant loss of NeuN immunoreactivity in the dentate gyrus and entorhinal cortex, while there were no changes in immunoreactivity in the CA3 pyramidal neurons [2].

Figure 5: Changes in optical density of the neurons in control animals compared to the lesioned animals.

2) The research team of Daly et al conducted the optokinetic response assay and the visual motor response assay in order to assess visual functioning in dye mutant zebrafish. The optokinetic response was meant to measure the ability to track objects by recording the number of saccadic eye movements exhibited by the larvae in response to rotating grated pattern stimulus. The 5 days post-fertilization dye zebrafish were shown to display a 98% reduction to optokinetic response with an average of 0.33 saccades per minute. This was in comparison to the unaffected sibling subjects that experienced an average of 21.6 saccades per minute [3].

Figure 6: Changes in the optokinetic response measured by the number of saccadic eye movements per minute in dye mutant zebrafish in comparison to the unaffected siblings.

The optokinetic response assay was followed by the visual motor response assay that was meant to quantify the locomotor activity of the larvae in response to sudden changes in lighting. The changes in lighting were defined as an ON response when the environment was illuminated, or an OFF response when the environment was darkened. These changes typically resulted in a startled response from the zebrafish that was characterized by an acute increase in locomotion. That being said, in comparison to the unaffected siblings, 5 days post-fertilization dye mutant zebrafish exhibited reduced visual motor response activity with decreased ON and OFF peak responses and no increase in light seeking behavior when in dark environments [3].

The dye mutants experienced a 79% reduction in MAX OFF visual motor responses and produced an average peak response of 0.055 ms.s. This was in comparison to the unaffected siblings that experienced a 0.259 ms/s OFF visual motor response. Additionally, the dye mutants displayed an 85% reduction in the MAX ON visual motor response, producing a peak average response of 0.054 ms/s. This was compared to the unaffected siblings that experienced a 0.354 ms/s ON visual motor response. Electroretinography was conducted following the optokinetic response and visual motor response testing in order to directly measure functioning of the outer retina. The dye mutant zebrafish exhibited a defective electroretinogram that displayed reduced or absent b-waves in response to 20 ms flashes of varying intensities of light [3].

Figure 7: Changes in C) average activity of OFF visual motor responses and D) average activity of ON visual motor responses in dye mutant zebrafish.

In addition to the behavioral analyses, the researchers utilized various histone deacetylase inhibitors (HDACi) to assess the preservation of retinal morphology. After treating the subjects with the HDACi mediators, preservation of the visual function was due to an increase in endogenous BDNF production that led to modulation of TrkB signaling. That being said, the dye mutant zebrafish were then treated with 10 uM of the BDNF mimetic 7,8-dihydroxyflavone hydrate which was found to enhance retinal morphology while not changing any of the gross morphological defects present.

The 10 uM dose of 7,8-dihydroxyflavone hydrate was also found to rescue visual functioning as measured by the optokinetic response assay and the visual motor response assay. The optokinetic response was shown to increase by 57.58 fold while the ON visual motor response increased by 3.89 fold and the OFF visual motor response increased by 3.33 fold. When 10 uM of 7,8-dihydroxyflavone hydrate was administered in combination with 500 nM ANA-12, the optokinetic response decreased by 71% while the ON visual motor response decreased by 58%, and the OFF visual motor response decreased by 48%. Overall, based on the findings presented the research team was able to conclude that BDNF-TrkB signaling regulates HDACi to rescue visual functioning [3].

Figure 8: Changes in K) optokinetic response and I) ON and OFF visual motor responses in dye mutants treated with 10 uM of 7,8-dihydroxyflavone hydrate

 

Disclaimer

**LAB USE ONLY**
*This information is for educational purposes only and does not constitute medical advice. THE PRODUCTS DESCRIBED HEREIN ARE FOR RESEARCH USE ONLY. All clinical research must be conducted with oversight from the appropriate Institutional Review Board (IRB). All preclinical research must be conducted with oversight from the appropriate Institutional Animal Care and Use Committee (IACUC) following the guidelines of the Animal Welfare Act (AWA).

 

Citations

[1] Liu X, Chan CB, Qi Q, et al. Optimization of a small tropomyosin-related kinase B (TrkB) agonist 7,8-dihydroxyflavone active in mouse models of depression. J Med Chem. 2012;55(19):8524-8537. doi:10.1021/jm301099x

[2] Castello NA, Nguyen MH, Tran JD, Cheng D, Green KN, LaFerla FM. 7,8-Dihydroxyflavone, a small molecule TrkB agonist, improves spatial memory and increases thin spine density in a mouse model of Alzheimer disease-like neuronal loss. PLoS One. 2014;9(3):e91453. Published 2014 Mar 10. doi:10.1371/journal.pone.0091453.

[3] Daly C, Shine L, Heffernan T, et al. A Brain-Derived Neurotrophic Factor Mimetic Is Sufficient to Restore Cone Photoreceptor Visual Function in an Inherited Blindness Model. Sci Rep. 2017;7(1):11320. Published 2017 Sep 12. doi:10.1038/s41598-017-11513-5

Mechanisms of Action

7,8-dihydroxyflavone hydrate (7,8-DHF) is a newly discovered flavonoid compound identified during a screening study aiming to find molecules with neurotrophic factors similar to that of brain-derived neurotrophic factor (BDNF). The results of the study reported that 7,8-DHF is a crucial activator of the BDNF signaling pathway. This leads to the prevention of apoptosis, increased neurogenesis, and an improvement in neuroprotection.

This initial study also found that 7,8-DHF acts as a direct ligand to tropomyosin-related kinase B (TrkB) receptors. In order to promote neuron growth and survival, TrkB is typically activated by BDNF. This leads experts to believe that not only does 7,8-DHF play a role in the BDNF signaling pathway, but the compound may be able to mimic the actions of BDNF and elicit the same expected outcomes via TrkB activation.

There has been a multitude of animal-based studies examining how 7,8-DHF is absorbed in the body. Poor absorption is a trait typically seen in flavonoids. In the case of 7,8-DHF, the compound is capable of oral absorption, however, it is metabolized so quickly it doesn’t make it to the bloodstream. Researchers Liu et. Al have been modifying the structure of the compound in an attempt to optimize its effects. Liu et. Al determined that these modifications have led to 7,8-DHF becoming orally bioavailable in mice, however, the compound is still subject to rapid, first-pass metabolism. Further research should be conducted in order to determine whether manipulation of various oral pharmacokinetic properties could be deemed beneficial (https://pubmed.ncbi.nlm.nih.gov/22984948/).

On the other hand, researchers Andero et. Al examined how 7,8-DHF was absorbed when peripherally injected rather than orally administered. 5 mg/kg doses of the compound were administered to mice; results of the study reported that TrkB receptors were activated. This suggests that 7,8-DHF is capable of effectively passing the blood-brain-barrier (BBB), a task BDNF is not capable of.

 

Effects of 7,8-DHF on Neurogenesis

As it was previously mentioned, 7,8-DHF has been shown to potentially increase neurogenesis and prevent apoptosis. Researchers have theorized that this is due to the compound’s interaction with TrkB receptors and BDNF, however, data is still mixed in regards to the exact mechanism of action and how heavily the compounds are related. A notable study conducted by Zhang et. Al reported that administration of 500 nM of 7,8-DHF in vitro led to an increase in dendritic length and synaptic size. Researchers Castello et. Al supported this conclusion, and further determined that 7,8-DHF may not lead to an increase in axon terminals, but rather promote growth of the density dendrites. It is important to know that growth of the density dendrites is directly related to expression of BDNF via 7,8-DHF-induced TrkB activation (https://pubmed.ncbi.nlm.nih.gov/24614170/).

The work of Castello et. Al was solidified by additional studies examining the efficacy of 7,8-DHF in cases of Alzheimer’s Disease. Mice subjected to scopolamine-induced Alzheimer’s were injected with doses of 1 mg/kg of 7,8-DHF. Results reported that the subjects experienced restoration in field excitatory postsynaptic potential to the same level as the control subjects. These results indicate an overall improvement in synaptic transmission. Further research conducted by Lin et. Al examined the effects of 7,8-DHF on a transgenic model of Alzheimer’s in mice where hippocampal cells are destroyed. The researchers were able to conclude that 5 mg/kg doses of the compound led to restoration of synaptic density in damaged neurons.

 

Effects of 7,8-DHF on Metabolism and Performance

Current research has found that 7,8-DHF is able to positively affect aspects of metabolic rate and physical performance through its relationship to BDNF and TrkB receptors. As it was previously mentioned, 7,8-DHF is able to activate and mimic both these crucial variables. Researchers have concluded that BDNF tends to lower food intake in rats by binding to the TrkB receptors, however, BDNF has a short half-life and cannot permeate the BBB. This led to the hypothesis that 7,8-DHF may be a better option for improving metabolism due to its similarity to BDNF and ability to pass the BBB. This idea is supported by the work of Chan et. Al. The researchers examined how daily administration of 7,8-DHF for 20 weeks affected obese mice and found that overall the compound is capable of preventing obesity.

In terms of physical performance, neurotrophins such as BDNF are known to be involved in muscle contraction through the activation of TrkB receptors that promote the release of acetylcholine (ACh). Since 7,8-DHF is closely related to both BDNF and TrkB receptors, researchers Mantilla et. Al determined how neuromuscular transmission was changed when 7,8-DHF was administered. Results reported that when the mice test subjects were given 10µM of 7,8-DHF, there was a 32% improvement in neuromuscular transmission. While these conclusions are promising, further research should be conducted in order to determine additional benefits (https://pubmed.ncbi.nlm.nih.gov/22246885/).

The nootropics sold by Umbrella Labs are sold for laboratory research only. The description above is not medical advice and is for informative purposes only.

7,8-Dihydroxyflavone Hydrate (DHT) is sold for laboratory research use only. Terms of sale apply. Not for human consumption, nor medical, veterinary, or household uses. Please familiarize yourself with our Terms & Conditions prior to ordering.

 

 

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