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Randomized Controlled Trial
. 2019 Aug;38(6):526-536.
doi: 10.1080/07315724.2018.1564088. Epub 2019 Jun 4.

Skin Transcriptome of Middle-Aged Women Supplemented With Natural Herbo-mineral Shilajit Shows Induction of Microvascular and Extracellular Matrix Mechanisms

Amitava Das  1   2 , Mohamed S El Masry  1   2   3 , Surya C Gnyawali  2 , Subhadip Ghatak  2   4 , Kanhaiya Singh  1   2 , Richard Stewart  2 , Madeline Lewis  2 , Abhijoy Saha  5 , Gayle Gordillo  4   6 , Savita Khanna  2   4
Affiliations
Randomized Controlled Trial

Skin Transcriptome of Middle-Aged Women Supplemented With Natural Herbo-mineral Shilajit Shows Induction of Microvascular and Extracellular Matrix Mechanisms

Amitava Das et al. J Am Coll Nutr. 2019 Aug.

Abstract

Objective: Shilajit is a pale-brown to blackish-brown organic mineral substance available from Himalayan rocks. We demonstrated that in type I obese humans, shilajit supplementation significantly upregulated extracellular matrix (ECM)-related genes in the skeletal muscle. Such an effect was highly synergistic with exercise. The present study (clinicaltrials.gov NCT02762032) aimed to evaluate the effects of shilajit supplementation on skin gene expression profile and microperfusion in healthy adult females. Methods: The study design comprised six total study visits including a baseline visit (V1) and a final 14-week visit (V6) following oral shilajit supplementation (125 or 250 mg bid). A skin biopsy of the left inner upper arm of each subject was collected at visit 2 and visit 6 for gene expression profiling using Affymetrix Clariom™ D Assay. Skin perfusion was determined by MATLAB processing of dermascopic images. Transcriptome data were normalized and subjected to statistical analysis. The differentially regulated genes were subjected to Ingenuity Pathway Analysis (IPA®). The expression of the differentially regulated genes identified by IPA® were verified using real-time polymerase chain reaction (RT-PCR). Results: Supplementation with shilajit for 14 weeks was not associated with any reported adverse effect within this period. At a higher dose (250 mg bid), shilajit improved skin perfusion when compared to baseline or the placebo. Pathway analysis identified shilajit-inducible genes relevant to endothelial cell migration, growth of blood vessels, and ECM which were validated by quantitative real-time polymerase chain reaction (RT-PCR) analysis. Conclusions: This work provides maiden evidence demonstrating that oral shilajit supplementation in adult healthy women induced genes relevant to endothelial cell migration and growth of blood vessels. Shilajit supplementation improved skin microperfusion.

Keywords: ECM; Shilajit; aging; dietary supplementation; skin perfusion.

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Figures

Figure 1.
Figure 1.
Shilajit improves skin microperfusion. A, Study design. B, Dermascopic images of the cheek. C, MATLAB multicolor coded dermascopic images. D, 3-D scatterplot of the Visible Bands of MATLAB processed dermascopic images. E, The sum of the area under the curve of red and green channels were plotted graphically. The intensity of the red and green channels was calculated from the multicolor images processed by MATLAB software from the raw dermascopic images. S125 represents shilajit 125 mg and S250 represents shilajit 250 mg. Data are mean ± SEM (n = 13). *p < 0.05 compared to the baseline visit. †p < 0.05 compared to placebo.
Figure 2.
Figure 2.
Heat map illustrating cluster of transcripts sensitive to shilajit supplementation (250 mg bid). Shilajit-sensitive transcripts were subjected to hierarchical clustering. A, Heat map illustrating cluster of transcripts that were upregulated upon shilajit supplementation. B, Heat map (top 100 candidates) demonstrating cluster of transcripts that were upregulated upon shilajit supplementation.
Figure 3.
Figure 3.
A and B, Ingenuity pathway analysis (IPA) showing that the supplementation of shilajit induces ECM-related genes.
Figure 4.
Figure 4.
RT-PCR validation of ECM-related genes following oral shilajit supplementation. Expression levels of selected genes identified by IPA were independently verified using quantitative real-time PCR. S125 represents shilajit 125 mg and S250 represents shilajit 250 mg. Data are mean ± SEM (n = 10–13). *p < 0.05 compared to visit 2. †p < 0.05 compared to placebo.
Figure 5.
Figure 5.
Ingenuity pathway analysis (IPA) showing that the supplementation of shilajit increases genes involved in the (A) movement of endothelial cells and growth of blood vessels through the (B) VEGFA and (C) TGFβ1 pathway.
Figure 6.
Figure 6.
RT-PCR validation of genes related to movement of endothelial cells following oral shilajit supplementation. Expression levels of selected genes identified by IPA were independently verified using quantitative real-time PCR. S125 represents shilajit 125 mg and S250 represents shilajit 250 mg. Data are mean ± SEM (n = 10–13). *p < 0.05 compared to visit 2. †p < 0.05 compared to placebo.
Figure 7.
Figure 7.
RT-PCR validation of genes related to growth of blood vessels following oral shilajit supplementation. Expression levels of selected genes identified by IPA were independently verified using quantitative real-time PCR. S125 represents shilajit 125 mg and S250 represents shilajit 250 mg. Data are mean ± SEM (n = 10–13). *p < 0.05 compared to visit 2. †p < 0.05 compared to placebo.
Figure 8.
Figure 8.
Shilajit is safe for facial skin. A, TEWL; B, hydration; C, elasticity (E); D, viscoelasticity (VE); and E, retention time was measured using Dermalab combo®. S125 represents shilajit 125 mg and S250 represents shilajit 250 mg. Data are mean ± SEM (n = 13–14).
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