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Alzheimer’s disease (AD) is a multifactorial neurodegenerative disorder without a cure, despite the enormous number of investigations and therapeutic approaches. AD is a consequence of microglial responses to “damage signals”, such as aggregated tau oligomers, which trigger a neuro-inflammatory reaction, promoting the misfolding of cytoskeleton structure. Since AD is the most prevalent cause of dementia in the elderly (>60 years old), new treatments are essential to improve the well-being of…
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Scaling the Andean Shilajit: A Novel Neuroprotective Agent for Alzheimer's Disease - PubMed

Scaling the Andean Shilajit: A Novel Neuroprotective Agent for Alzheimer’s Disease

Víctor Andrade et al. Pharmaceuticals (Basel). .

Abstract

Alzheimer’s disease (AD) is a multifactorial neurodegenerative disorder without a cure, despite the enormous number of investigations and therapeutic approaches. AD is a consequence of microglial responses to “damage signals”, such as aggregated tau oligomers, which trigger a neuro-inflammatory reaction, promoting the misfolding of cytoskeleton structure. Since AD is the most prevalent cause of dementia in the elderly (>60 years old), new treatments are essential to improve the well-being of affected subjects. The pharmaceutical industry has not developed new drugs with efficacy for controlling AD. In this context, major attention has been given to nutraceuticals and novel bioactive compounds, such as molecules from the Andean Shilajit (AnSh), obtained from the Andes of Chile. Primary cultures of rat hippocampal neurons and mouse neuroblastoma cells were evaluated to examine the functional and neuroprotective role of different AnSh fractions. Our findings show that AnSh fractions increase the number and length of neuronal processes at a differential dose. All fractions were viable in neurons. The AnSh fractions inhibit tau self-aggregation after 10 days of treatment. Finally, we identified two candidate molecules in M3 fractions assayed by UPLC/MS. Our research points to a novel AnSh-derived fraction that is helpful in AD. Intensive work toward elucidation of the molecular mechanisms is being carried out. AnSh is an alternative for AD treatment or as a coadjuvant for an effective treatment.

Keywords: Alzheimer’s disease (AD); Andean Shilajit (AnSh); bioactive fractions; molecular networks; neuroprotector; prevalent neurological disorders.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1

Main fractions extracted from the Andean Shilajit. The diagram presents the polarity-based fractionation process for the M1, M2, M3, and M4 fractions.

Figure 2
Figure 2

N2a cells are viable after treatment with the different fractions of Andean Shilajit. Concentrations from 0.1 μg/mL to 100 μg/mL of each fraction of the AnSh (n = 4–5) were tested in N2a cells. Only high concentrations (50 and 100 μg/mL) of the M2 fraction were toxic to cells. ANOVA test, factor followed by Tukey’s post hoc test. *** = p-value < 0.001.

Figure 3
Figure 3

Immunohistochemical staining of N2a cells treated with M1, M2, M3, and M4 fractions for 24 h. Immunofluorescence assays representing the control condition (A). Protein markers used in the experiment are shown as follows: α-Tubulin: microtubule-associated protein marker; Phalloidin: β-actin protein marker; Topro: cell core, and merge of all the channels involved. (B) Representative images of N2a cells treated with 0.5 μg/mL, 1.0 μg/mL, 5.0 μg/mL, and 25.0 μg/mL of AnSh fractions.

Figure 4
Figure 4

Effect of AnSh fractions on the length of neurites in N2a cells. A wide range of concentrations of AnSh fractions morphologically modifies N2a cells. Concentrations from 0.5 μg/mL to 25 μg/mL of each AnSh fraction in N2a cells (n = 4–5), and up to 5 μg/mL of fulvic acid and total Andean Shilajit (n = 3) were tested. The values represent the standard error. ANOVA test, followed by Tukey’s post hoc test. * = p value < 0.05, ** = p value < 0.01, *** = p value < 0.001.

Figure 5
Figure 5

Effect of AnSh fractions on the number of neuritogenic processes (number of neurites) in N2a cells. Concentrations from 0.5 μg/mL to 25 μg/mL of each AnSh fraction (n = 4–5) in N2a cells, and up to 5 μg/mL of fulvic acid and total Andean Shilajit (n = 3) were tested. The values represent the standard error. ANOVA test, followed by Tukey’s post hoc test * = p value < 0.05.

Figure 6
Figure 6

Representative morphology images of N2a cells after treatment with different concentrations of the M3 subfractions. Immunofluorescence assays represent different assay conditions. (A) Control condition. Protein markers used in the experiment are shown as follows: α-Tubulin: microtubule-associated protein marker; Phalloidin: β-actin protein marker; Topro: cell core, and merge of all the channels involved. (B) The representative images of N2a cells present their morphological changes after being treated with 0.5 μg/mL, 1.0 μg/mL, and 5.0 μg/mL of the M3 subfractions for 24 h.

Figure 7
Figure 7

Subfractions of M3 modulate N2a morphology at a wide range of concentrations. Concentrations from 0.1 μg/mL to 5.0 μg/mL of each subfraction of M3 were tested in N2a cells (n = 3). All concentrations of Precipitate M3 subfraction elicited morphological changes (length and the number of neurites) in N2a cells after 24 h. Only 5.0 μg/mL of Polar M3 increased the length of neurites. Values represent the standard error. ANOVA test, factor followed by post hoc Tukey test. * = p-value < 0.05, *** = p-value < 0.001.

Figure 8
Figure 8

Representative morphology images of RHN cells after treatment with different concentrations of the Precipitate M3 subfraction or BrainUp-10®. Immunofluorescence assays represent different assay conditions. Control condition. Protein markers used in the experiment are shown as follows: Phalloidin: β-actin protein marker; α-Tubulin: microtubule-associated protein marker; Topro: cell core and merge of all the channels involved. The representative images of RHN present morphological changes after being treated with 0.5 μg/and mL; 1.0 μg/mL, 5.0 μg/mL of the Precipitate M3 subfraction or BrainUp-10® for 24 h. DMSO is shown as the vehicle solution control.

Figure 9
Figure 9

Precipitate M3 subfraction or BrainUp-10® elicited RHN cell morphological changes at a wide range of concentrations. Concentrations from 0.5 μg/mL to 5.0 μg/mL of both the Precipitate M3 subfraction and BrainUp10® were tested in RHN cells (n = 3). Concentrations of 0.5 and 5.0 μg/mL of Precipitate M3 subfraction resulted in an increased neurite length in RHN; meanwhile, the same effect was seen with 0.5 and 1.0 μg/mL of BrainUp-10®. Values represent the standard error. ANOVA test, factor followed by post hoc Tukey test. * = p-value < 0.05, *** = p-value < 0.001.

Figure 10
Figure 10

Representative effect of AnSh fractions on the aggregation in vitro of recombinant hTau 40 protein. Concentrations of 5 μg/mL of each AnSh M3 subfraction, M2, and M3 fractions (individually or in combination), fulvic acid (FA), Andean Shilajit (AnSh), and BrainUp-10® were tested (n = 1). (A) Representative tracing curves of aggregation of hTau 40 during 10 days of registration, with different treatments performed through Thioflavin S (ThS) fluorescence assay. (B) Representative images acquired by electron microscopy (magnification 25,000× g) of hTau 40 aggregation in the presence (+Hep, maximum) and absence (−Hep, minimum) of Heparin. (C) Inhibition percentage of hTau 40 aggregation obtained with different treatments. Highlighted are the values with the highest inhibition percentage.

Figure 11
Figure 11

Main compounds characterized by UPLC/MS in the M3 fraction precipitate. The chemical identities of the dipeptide (A) and ASM3 (B) were assigned based on the chromatographic profiles of the UV and MS spectra. Records were evaluated at 35.0 eV.

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Scaling the Andean Shilajit: A Novel Neuroprotective Agent for Alzheimer's Disease – PubMed