Description

9-Methyl-β-carboline (9-Me-BC) Nootropic Liquid

 

 

 

CAS Number	2521-07-5
Other Names	2521-07-5, 9-methyl-9h-pyrido[3,4-b]indole, GC837J2CCJ
IUPAC Name	9-methylpyrido[3,4-b]indole
Molecular Formula	C₁₂H₁₀N₂
Molecular Weight	182.22
Purity	≥99% Pure (LC-MS)
Liquid Availability	30mL liquid (50mg/mL, 1500mg bottle)
Powder Availability	1 gram, 5 grams, 60 capsules (20mg/capsule, 1200mg bottle)
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 9-Me-BC?

9-Methyl-𝛃-Carboline (9-Me-BC) is a potent nootropic that has shown promise in improving wakefulness, alertness, cognition, and mood. 9-Me-BC works by regulating the serotonergic and dopaminergic centers of the brain to decrease the reuptake of the two compounds. Evidence has shown that the increased levels of dopamine and serotonin help to greatly improve cognition and energy levels. The primary active compound of 9-Me-BC is the 𝛃-Carboline. 𝛃-Carboline are alkaloids found in various insects, plants, mammals, and marine animals, and are known for its beneficial biochemical and pharmacological effects. 𝛃-Carboline compounds have been shown to insert themselves into DNA, interact with 5-hydroxy serotonin and benzodiazepine receptors, and inhibit the actions of CDK, topoisomerase, and monoamine oxidase. These properties indicate that the compounds have strong antioxidant and anti-inflammatory effects [1].

 

Main Research Findings

1) 9-Me-BC has the potential to act as an anti-Parkinson’s Disease medication as the nootropic interferes with the pathogenic factors that facilitate degenerative processes in the substantia nigra.

2) Administration of 9-Me-BC has been shown to inhibit activity of monoamine oxidase while stimulating the expression of neurotrophic factors by astrocytes.

 

Selected Data

1) The research team of Polanski et al. examined the inflammatory processes associated with the activation of microglia and degeneration in the substantia nigra. For the purpose of this study pregnant C57B1/6 mice between 3 and 6 months old were utilized. On the 14th gestation day the animals were euthanized while dopaminergic cell cultures were prepared and embryonic mesencephala were dissected. 2 mL of trypsin-solution, 3 mL of Hank’s balanced salt solution, and 100 ul DNase I was added to the samples that were then incubated in a water bath 2 mL of basic medium was added and the tissue sample was centrifuged and the supernatant was discarded. 3 mL of basic medium was supplemented with 60 uL of DNase 1 following trituration. ½ of the medium was changed on the first day in vitro while ⅔ of the medium was changed on day 3 in vitro. On the 5th day in vitro the medium was replaced with serum-free DMEM that was used for feeding starting at day 6 in vitro and replaced every other day [2].

9-Me-BC was prepared as a 1 mM stock solution that was diluted in a B27-supplemented medium. 1 hour before treatment with 9-Me-BC the solution was incubated with 100 uM of tyrphostin or 100 uM of genistein, and 15 minutes before treatment with 9-Me-BC incubation with 3 um GBR12909 began. The toxins used were prepared as 1 mM stock solutions that were diluted in a B27-supplemented medium. The cultures were treated with 2,9-dime-BC, MPP, LPS, or protein on the 10th day in vitro in order to induce acute injury. In order to induce chronic injury rotenone was added to the cultures from 6-12 days in vitro, every other day.

Immunocytochemical staining was used to identify microglia. The samples were then rinsed with DPBS followed by precooled with Accustain and washing with PBS. Cultures were then incubated to develop a reaction product. The resulting cells were counted using an inverted microscope in order to identify the average number of TH immunoreactive cells. After TH cell cultures were immunochemical stained, 10 of the cells were randomly allocated for additional monitoring in order to quantify neurite growth. Primary neurons were defined as every neurite that grew out of the soma while secondary neurites were defined as neurites originating from the first branching point of primary neurites [2].

Cell injury was determined by measuring the release of lactate dehydrogenase into the medium. Levels of LDH were determined through the use of the LDH CYtotoxicity Detection Kit. From there EdU was incorporated into the samples in order to quantify cell proliferation in the cultures. The cells were incubated for 4 hours with 10 uM of EdU, 12 hours with 5 uM of EdU, or 24 hours with 3 uM of EdU, followed by 48 hours of treatment with 9-Me-BC. From there the cell cultures were immunofluorescently stained for TH in order to determine if TH and EdU were co-localized. Protein concentrations were then determined using fluorescence technology after the cell cultures were rinsed with PBS and harvested in 100 uL of LDS and sample buffer [2].

The harvested protein samples were then incubated with 5 uL of NuPAGE LDS sample buffer in order to denature the cultures. The proteins were separated and the membranes were incubated with TH antibody in order to detect changes in the samples through the use of a chemiluminescent kit. The resulting images were stored for further analysis. Total RNA was also isolated in order to measure RNA concentration. The cells were rinsed with PBS and stabilized, followed by mixing with lysate buffer and homogenization using a spin column. Using an RNA assay kit, both concentration and purity of RNA in the samples was determined. Finally, inflammatory cytokines and receptors were measured, purified, and hybridized. Chemiluminescent images were obtained for further analysis [2].

2) The research team of Keller et al examine the neuroproliferative, neuroregenerative, and neuroprotective role of 9-Me-BC and how they are affected by the presence of astrocytes. For the purpose of this study, pregnant C57BL/6 mice between 3 and 6 months of age were euthanized via asphyxiation on gestation day 14, followed by the collection and preparation of dopaminergic cell cultures. On the first day in vitro, 1/2 of the basic medium was changed, and on the third day in vitro ⅔ of the basic medium was changed. On day 5 in vitro, ½ of the basic medium was replaced with serum-free Dulbecco’s modified Eagle’s medium (DMEM) containing 2% B-27. serum-free supplemented DMEM was also used for feeding starting on day 6 in vitro [3].

Once the animals were born they were euthanized and their brains were dissected in order to abrade the cortices of the right and left hemispheres. Collected samples were transferred in Dulbecco’s phosphate-buffered saline (DPBS). The meninges were removed and the cortices were further dissected into smaller pieces for further preparation and examination. Dissociated cell cultures were placed in a basic medium. ½ of the medium was changed on the first day in vitro, ⅔ was changed on third day in vitro, ½ of the medium was changed on the fifth day in vitro and on day six and day eight in vitro all of the medium was replaced. Prior to changing the medium on day eight in vitro, the cultures were shaken to remove non-astrocytic cells. Following incubation with trypsin solution and centrifugation, the astrocytes cultures were plated and placed in basic medium [3].

Cell cultures were then rinsed with DPBS and fixed in Histochoice Tissue Fixative for 30 minutes. This was followed by permeabilization with 0.4% Triton X-100 and incubation with anti-TH antibody. Biotinylated secondary antibody was then used to treat the cultures for an hour before the final addition of avidin-biotin-horseradish peroxidase complex. The number of cells were counted and images were obtained utilizing a computer driven digital camera and inverted microscope. Next, the cells wre treated with Disprocynium 24 (D24), a compound that acts as an inhibitor of organic cation transporters 1, 2, 3 (OCT1, OCT2, OCT3). Incubation with D24 began 30 minutes prior to 48 hours of treatment with 9-Me-BC. LY294002 hydrochloride and sulpiride were administered to the cell cultures next to inhibit PI3K pathways. Cell cultures were incubated with 10 uM LY294002 hydrochloride or 20 uM sulpiride 30 minutes before 9-Me-BC was added to the cultures [3].

Adenylate kinase activity was measured as a marker of cell injury using a bioassay kit. 20 uL of the supernatant collected from the cultures on day 14 in vitro. The release of lactate dehydrogenase into the medium was also observed as a marker of cell injury and was assessed spectrophotometrically. 10 mg/kg of Hoechst 33342 in H2O and 1 mg/ml of PI in H2O were prepared and incubated for 5 minutes with the cell cultures in serum-free supplemented DMEM medium. Apoptotic and necrotic cells were detected by measuring the uptake of PI into the cell nuclei, however, it was not possible to differentiate between apoptosis and necrosis. PI was visualized using the TRITC filter and blue fluorescence with the DAPI filter.

Astrocyte viability was measured on the 14th day in vitro using a cell viability assay kit. Cell proliferation was then quantified through the incorporation of BrdU and measurement using cell proliferation ELISA BrdU. The BrdU labeling solution was added to the cell cultures for incubation, followed by treatment with FixDennat and incubation Anti-BrdU peroxidase working solution. Finally, monoamine oxidase activity was determined by preparing a solution of 9-Me-BC in the MAO reaction buffer for MAO-A activity while an additional 10% DMSO was added for MAO-B activity. The compounds were incubated for 1 hour and the luciferase reaction was halted after 20 minutes [3].

 

Discussion

1) The results of the study conducted by the research team of Polanski et al found that when primary dopaminergic cultures were treated with 9-Me-BC for 48 hours, there was a significant increase in the number of THir neurons in a concentration-dependent manner. The number of neurons increased by 48% when the compound was administered in concentrations of 90 uM. These results were confirmed by the completion of western blot analysis of TH expression. As it was previously mentioned, primary neurons were defined as every neurite that grew out of the soma while secondary neurites were defined as neurites originating from the first branching point of primary neurites. The number of primary neurites from THir neurons increased from 4.75 to 6.7 and the number of secondary neurites increased from 3.99 to 5.51. Additionally, when the cell cultures were co-treated with DAT-inhibitor GBR12909 there was a diminishing effect elicited on the 9-Me-BC-induced stimulation of THir neurons. However, there was still a significant increase in the number of primary and secondary neurites from 4.74 to 6.23 and 3.85 to 5.03, respectively, indicating that the DAT-inhibitor did not affect neurite outgrowth [2].

Figure 4: Changes in concentration-dependent stimulation of THir neurons and (a) primary and secondary neurites; (b) after treatment with 9-Me-BC; (c) after co-treatment with GBR12909, and d) the relative protein expression of TH after treatment with 9-Me-BC.

After identifying the effects of 9-Me-BC on stimulation of THir neurons, the research investigated additional THir neuronal cultures derived from proliferative cells and treated with nucleoside analog, EdU, for either 4 hours, 12 hours, or 24 hours. It was revealed that even after 24 hours of incubation none of the THir neurons tested as EdU-positive. In a similar manner, neurons treated with 90 uM of 9-Me-BC for 48 hours did not result in the incorporation of EdU in THir neuronal cultures. These findings support the hypothesis proposed by the research team that administration of 9-Me-BC only affects pre-existing dopaminergic neurons.

In order to further examine whether 9-Me-BC affects pre-existing and “silent” dopaminergic neurons that do not express TH, cultures were stained for dopa decarboxylase immunoreactive neurons following 48 hours of incubation with 90 uM 9-Me-BC. There was a significant increase in the number of THir neurons, and each neuron was shown to be dopa decarboxylase immunoreactive. In the control groups that were not treated with 9-Me-BC, levels of dopa decarboxylase immunoreactive neurons were 50% more than THir neurons. Compared to the groups treated with 90 uM of 9-Me-BC, were levels of dopa decarboxylase immunoreactive neurons were 23% more than THir neurons. Furthermore, when treatment with 9-Me-BC was stopped, the number of THir neurons decreased to 8-10% above baseline values. Similar results were seen with the number of primary and secondary neurites [2].

9-Me-BC was also assessed for its neuroprotective and anti-inflammatory effects against various dopaminergic neurotoxins. The cultures were pretreated for 48 hours with 90 uM of 9-Me-BC followed by incubation with mitochondrial inhibitors Mpp+, 2,9-dime-BC+, rotenone, or inflammation-inducing LPS for 48 hours. Lipopolysaccharide was shown to decrease the number of THir neurons by 37% while the number of microglia increased by 300% and the release of LDH increased by 84%. When the cell cultures were co=treated with LPS and 9-Me-BC the number of THir neurons increased by 16%. This was compared to the untreated controls that experienced a significant reduction in microglia and LDH release compared to LPS controls. These results indicate that reduction of a pro-inflammatory environment caused by the proliferation of microglia may contribute to the neuroprotective effects elicited by 9-Me-BC [2].

The dopaminergic model toxin MPP+ was shown to decrease the number of THir neurons by 72% while increasing the number of microglia cells by 193% and LDH release by 100%. Co-treatment and pre-treatment with 9-Me-BC was shown to reduce microglia proliferation and release of LDH. These findings suggest that the inhibitory effects of MPP+ on the mitochondrial respiratory chain outweighs the potential protective effects caused by reducing microglia proliferation. Furthermore, rotenone was found to decrease the number of THir neurons by 68% while increasing microglia proliferation by 209% and LDH release by 103%. Co-treatment and pretreatment with 9-Me-BC resulted in further deterioration of THir neurons but also reduced proliferation of microglia.

Finally, 2,9-dime-BC+ was shown to decrease the number of THir neurons by 51% while microglia proliferation and LDH release was increased by 298% and 130%, respectively. Co-treatment with 9-Me-BC resulted in THir neurons being protected from deterioration while less protection was provided to the cell cultures when they were pre-treated with 9-Me-BC. Both co-treatment and pre-treatment with 9-Me-BC resulted in a significant decrease in microglia proliferation and LDH release. These findings suggest that co-treatment with the nootropic was more effective at combating neurotoxicity elicited by 2,9-dime-BC+ [2].

Figure 5: Changes in (a) % of THir neurons, (b) % of microglia, and (c) LDH release after LPS administration. Changes in (d) % of THir neurons, (e) % of microglia, and (f) LDH release after MPP+ administration. Changes in (g) % of THir neurons, (h) % of microglia, and (i) LDH release after rotenone administration. Changes in (j) % of THir neurons, (k) % of microglia, and (l) LDH release after 2,9-dime-BC+ administration.

2) The results of the study conducted by Keller et al found that when mesencephalic dopaminergic cell cultures were treated with 9-Me-BC for 48 hours there was a concentration-dependent increase in the number of TH+ neurons, with maximum stimulation noted after treatment with 90 uM of 9-Me-BC. Cell cultures were also co-treated with sulpiride and 9-Me-BC in order to see if the stimulatory effect on TH+ neurons was mediated by dopamine receptors 2 and 3. It was found that incubation with 20 uM sulpiride for 48 hours did not result in a decreased number of TH_ neurons. Additionally, when treated with 70 uM of 9-Me-BC there was a increase in the number of TH+ neurons that was not significantly affected when the cultures were co-treated with 20 uM of sulpiride. These findings indicate that the stimulatory effects of 9-Me-BC are independent of dopamine receptors [3].

Next, the researchers assessed whether the survival of TH+ cells were influenced by astrocyte growth. Treatment with 9-Me-BC was shown to increase the levels of TH+ neurons, however, in comparison to the mesencephalic dopaminergic cultures lower concentrations of the nootropic were necessary to exert an increase in neurons. Additionally when astrocyte-depleted cell cultures were treated with 150 uM of 9-Me-BC, the number of TH+ neurons was shown to decrease by 50%. In mesencephalic dopaminergic cultures, the same treatment was found to only result in a slight reduction of dopaminergic neurons. The research team also noted that there was an impaired morphology of TH+ neurons that was observed in astrocyte-depleted cultures that improved significantly following treatment with the nootropic compound [3].

Figure 6: Changes in levels of TH+ neurons a) in dopaminergic cell cultures, b) when co-treated with sulpiride, and c) in astrocyte depleted cultures.

When assessing the anti-proliferative properties of 9-Me-BC, it was found that treatment did not result in increased adenylate kinase concentration. This indicated that the nootropic does not elicit any toxic effects on cortical astrocytes. Astrocyte cultures also showed increased viability by 12% and 20% following incubation with 90 uM and 150 uM of 9-Me-BC, respectively. These results were confirmed by MTT and measurements of PI and Hoechst 33342 incorporation. Furthermore, incubation of astrocyte cultures with the 9-Me-BC were shown to decrease the number of dead cells by 21% and LDH release by 73%. BrdU incorporation was also found to reduce significantly following incubation with 90 uM and 150 uM of the nootropic by 39% and 71%, respectively. These findings suggest that 9-Me-BC has antiproliferative effects in cortical astrocytes [3].

Figure 7: Changes in a) adenylate kinase activity, b) viability of cortisol astrocyte, c) cell mortality, d) LDH release, and e) BrdU incorporation following treatment with 9-Me-BC, as well as f) BrdU incorporation when co-treated with D24.

 

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] Gruss M, Appenroth D, Flubacher A, Enzensperger C, Bock J, Fleck C, Gille G, Braun K. 9-Methyl-β-carboline-induced cognitive enhancement is associated with elevated hippocampal dopamine levels and dendritic and synaptic proliferation. J Neurochem. 2012 Jun;121(6):924-31. doi: 10.1111/j.1471-4159.2012.07713.x. PMID: 22380576.

[2] Polanski W, Reichmann H, Gille G. Stimulation, protection and regeneration of dopaminergic neurons by 9-methyl-β-carboline: a new anti-Parkinson drug? Expert Rev Neurother. 2011 Jun;11(6):845-60. doi: 10.1586/ern.11.1. PMID: 21651332.

[3] Keller S, Polanski WH, Enzensperger C, Reichmann H, Hermann A, Gille G. 9-Methyl-β-carboline inhibits monoamine oxidase activity and stimulates the expression of neurotrophic factors by astrocytes. J Neural Transm (Vienna). 2020 Jul;127(7):999-1012. doi: 10.1007/s00702-020-02189-9. Epub 2020 Apr 13. Erratum in: J Neural Transm (Vienna). 2022 Jan;129(1):125. doi: 10.1007/s00702-021-02433-w. PMID: 32285253; PMCID: PMC8592951.

What is 9-ME-BC?

9-Methyl-𝛃-Carboline (9-Me-BC) is a potent nootropic that has shown promise in improving wakefulness, alertness, cognition, and mood. 9-Me-BC works by regulating the serotonergic and dopaminergic centers of the brain to decrease the reuptake of the two compounds. Evidence has shown that the increased levels of dopamine and serotonin help to greatly improve cognition and energy levels.

The primary active compound of 9-Me-BC is the 𝛃-Carboline. 𝛃-Carboline are alkaloids found in various insects, plants, mammals, and marine animals, and are known for its beneficial biochemical and pharmacological effects. 𝛃-Carboline compounds have been shown to insert themselves into DNA, interact with 5-hydroxy serotonin and benzodiazepine receptors, and inhibit the actions of CDK, topoisomerase, and monoamine oxidase. These properties indicate that the compounds have strong antioxidant and anti-inflammatory effects [1].

 

Main Research Findings

1. 9-Me-BC has been shown to enhance cognition by regulation of the dopaminergic system. The increased dopamine levels are strongly correlated to proliferation of dendrites and synapses

2. Additional research conducted on this compound has found that due the regulation of dopamine that occurs, 9-Me-BC could potentially be considered as a treatment for Parkinson’s Disease.

 

Selected Data

1. 7-week-old female Wistar rats were split into 4 experimental groups: non-injected animals, vehicle-injected animals, animals treated with 9-Me-BC for 5 days, and animals treated with 9-Me-BC for 10 days. The radial maze (RAM) test was used in order to measure changes in spatial learning. The first RAM test was conducted on day 0 before any treatment began while all following tests took place everyday approximately 3 hours after treatment [2].

2. Researchers Polanski et. Al have determined that 9-Me-BC has different beneficial properties that could support the claim that the compound is an effective treatment for Parkinson’s. This theory is based on the observed activity between 9-Me-BC and variables such as tyrosine hydroxylase and various immunoreactive neurons [3]

 

Discussion

1. By decreasing the uptake of dopamine, which is known to be an important neurotransmitter, the compound is able to “relay its message” but is not reabsorbed by the synapse. This indicates that dopamine is active and present in the neuronal receptors for a longer period of time, thus leading to cognitive benefits. Results of the study conducted by found that after 7 days of treatment, rats given 9-Me-B for 10 days were already successful in completing the RAM test. However, there was no difference in the amount of errors made by the rats treated with a vehicle and the rats treated with 9-Me-BC for 5 days.

Figure 1: Decrease in errors on RAM test with administration of 9-Me-BC

The hippocampal tissue of the rats were examined further in order to measure changes in hippocampal dopamine levels and proliferation of synapses and dendrites. Again, there was no difference in the hippocampal dopamine levels of the rats administered 9-Me-BC for 5 days and to those given the vehicle. However, the rats given 9-Me-BC for 10 days saw a significant increase in hippocampal dopamine levels, almost double the levels of the non-injected animals.

Figure 2: Changes in dopamine levels based on the different experimental variables

It is important to note that when observing synaptic and dendritic proliferation, the only data compared was from the rats injected with a vehicle and those receiving 9-Me-BC for 10 days. That being said, there was drastic growth found in the synapses and dendrites of the rats receiving 9-Me-BC. The most significant changes were seen at 60 μm, 110 μm, and 160 μm [2].

Figure 3: Changes in the dendrites after administration of 9-Me-BC

2. 9-Me-BC is capable of increasing the release of tyrosine hydroxylase and its various transcription factors in immunoreactive neurons. This ultimately has a stimulatory effect on neurons of the dopaminergic system. Furthermore, 9-Me-BC has been shown to induce gene expression of different neurotrophic factors while also decreasing the rate of apoptosis cell signaling. This indicates that the compound has both protective and regenerative properties towards the dopaminergic neurons. 9-Me-BC can combat inflammation by inhibiting monoamine oxidase and the proliferation of microglia through the reduction of chemotactic cytokines.

Both the protection of the dopaminergic neurons and the overall anti-inflammatory properties the compound promotes is what leads researchers to believe 9-Me-BC could be used as a treatment for Parkinson’s Disease, considering dopamine and inflammation play a key role in the disease. However, further research is currently being conducted to hone in on the properties of the nootropic compound [3]

 

Conclusions

**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] Cao R, Peng W, Wang Z, Xu A. beta-Carboline alkaloids: biochemical and pharmacological functions. Curr Med Chem. 2007;14(4):479-500. doi: 10.2174/092986707779940998. PMID: 17305548.

[2] Gruss M, Appenroth D, Flubacher A, Enzensperger C, Bock J, Fleck C, Gille G, Braun K. 9-Methyl-β-carboline-induced cognitive enhancement is associated with elevated hippocampal dopamine levels and dendritic and synaptic proliferation. J Neurochem. 2012 Jun;121(6):924-31. doi: 10.1111/j.1471-4159.2012.07713.x. PMID: 22380576.

[3] Polanski W, Reichmann H, Gille G. Stimulation, protection and regeneration of dopaminergic neurons by 9-methyl-β-carboline: a new anti-Parkinson drug? Expert Rev Neurother. 2011 Jun;11(6):845-60. doi: 10.1586/ern.11.1. PMID: 21651332.

9-Methyl-β-carboline (9-Me-BC) 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.

*Note: A slight coloration shade may occur in between different batches; this is a normal occurrence for this research compound.

 

 

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