Interrogating the molecular responses of nutraceuticals to gain insights into their health benefits

Given increases in average human lifespan and changing population demographics, efforts to ensure healthy ageing are increasingly important not only for the quality of life of us as individuals as we age, but also for society as a whole in having to bear the costs to care for an ageing population. At Sibelius we believe that – alongside other components of a healthy lifestyle such as diet and exercise – nutraceuticals can make an important contribution towards supporting healthy ageing.

As we discussed in our previous article in Nutraceuticals Now1, short-lived model organisms such as the nematode worm Caenorhabditis elegans (Figure 1A), provide us with the ability to screen and identify ingredients that extend lifespan through action on core pathways of cellular ageing, which are conserved across species from single- celled organisms such as yeast all the way through to man. Such lifespan extending ingredients have the potential to impart benefits for healthy ageing and age-related diseases in humans. However, even in such model systems, treatments that extend lifespan do not always result in improvements to healthy ageing2, and it is increasingly being realised that there is a need to extend the quality of life (health-span) not simply the quantity of life (lifespan)3. Investigating the mechanisms by which a nutraceutical induces a biological response is therefore crucial to understanding whether it might contribute towards healthy ageing, and in particular which age- related conditions it may have the greatest benefits on.

Figure 1A – Increase of C. elegans lifespan by Sibelius:™Sage
Picture of the circa 1mm long nematode worm C. elegans

The first botanical extract that Sibelius has brought to market is Sibelius™:Sage, a proprietary cultivar of sage grown in the UK. Sibelius™:Sage shows increases in lifespan of around 10-15% in C. elegans (Figure 1B). It also shows significant improvements in cognitive performance in healthy seniors in clinical testing of humans4. One of the activities of Sibelius™:Sage that is likely to contribute to this improved cognitive performance is the inhibition of acetylcholinesterase (AChE)4, an enzyme which breaks down esters of the neurotransmitter acetylcholine, which plays an important role in the formation of memories5. This anti-AChE activity is shared by several drugs used in the treatment of early symptoms of Alzheimer’s Disease, including donepezil6, and likely contributes towards Sibelius™:Sage’s anti-ageing effects, since C. elegans worms treated with donepezil also show significant extension of lifespan of close to 10% on our Chronoscreen™ platform. However, the question remains as to what other activities Sibelius™:Sage has that contribute towards its cognitive and anti-ageing benefits (as well as the other health benefits related to traditional applications of sage as a herbal medicine).

Figure 1B – Increase of C. elegans lifespan by Sibelius:™Sage
Example of a survival curve for C. elegans worms, showing the percentage of the population of the still alive over 25 days of analysis.  Worms treated with Sibelius™:Sage (Green line) show an increase in median life expectancy of 10-15% versus control worms (Red line) on Sibelius’s Chronoscreen™ lifespan assay.

Measuring the expression of genes to identify those that are induced or repressed in response to different treatments can provide very valuable insights into how they are imparting biological effects. To further our understanding of the action of Sibelius™:Sage, we tested the gene expression responses of young adult C. elegans worms treated with the herbal extract. As a comparison we also tested gene expression responses of worms treated with donepezil and another herbal extract that shows lifespan extending activity, but has no evidence of anti-AChE activity.

A subset of the genes that we identified that respond to Sibelius™:Sage also responded to donepezil, which is consistent with the treatments sharing some similarity in action (Figure 2A). Likewise, a distinct subset of genes that responded to Sibelius™:Sage, also responded to treatment with the other lifespan extending herbal extract, again consistent with them sharing some similarity in action. As noted above, this other herbal extract has no reported anti-AChE activity and – as would thus be expected – does not show any genes responding in common with donepezil (Figure 2A).

Figure 2A – Investigating the biological effects of anti-ageing treatments
Gene expression levels were measured in young adult worms using the Affymetrix GeneChip™ C. elegans Gene 1.1 ST Array, which provides genome-scale coverage. Venn diagram summarising the overlap of genes showing expression responses to Sibelius™:Sage, donepezil and Herbal Extract X (another herbal extract that shows life-span extension on Chronoscreen™.
Heatmap summarising the gene expression changes for all treatments for the set of differentially expressed genes from panel A.

For many genes the function – or role – that they play in a biological system is known. Both Sibelius™:Sage and donepezil treatments caused an increase in expression of a set of genes that are known as cytochrome P450s (Figure 2B). These are a large family of genes found in all kingdoms of life, and play an important role in the metabolism of endogenous and exogenous substrates, such as pharmaceutical drugs in humans7. The genes up-regulated by Sibelius™:Sage and donepezil in C. elegans map to the same set of orthologous (related) cytochrome P450s in humans. These cytochrome P450s are not up-regulated in response to treatment with the other herbal extract (Figure 2B), so it seems likely that they are part of the response to the molecules present in Sibelius™:Sage as well as donepezil that contribute towards their anti-AChE activity.

Figure 2B – Investigating the biological effects of anti-ageing treatments
Each row represents expression for a single gene and is presented as the Log2 fold change versus the control for each of the treatments, with down-regulated genes presented in blue and up-regulated genes presented in yellow (see inset key for scale). Two groups of genes with similar expression patterns and similar biological roles are indicated by black bars to the right of the heatmap. Dendograms summarise the similarity of treatments (above) and genes (left) based on the expression patterns of the set of genes presented.

Similarly, the sets of genes that are induced in both Sibelius™:Sage and the other herbal extract, but which are not induced in response to donepezil, can be identified. These include a group of genes that are related to lipid metabolism or insulin signalling (Figure 2B), suggesting that both Sibelius™:Sage and the other herbal extract may impart effects on these areas of metabolism. Sage has previously been linked with hypoglycaemic effects in rats in in vivo studies8,9, which also showed beneficial effects on inflammation markers in serum9. As such it is possible that Sibelius™:Sage, as well as the other herbal extract, will have beneficial effects on human metabolic health, obesity, and associated inflammation. These benefits could also contribute to cognitive health by reducing an individual’s long-term risks of developing cognitive decline and dementia10,11.

As well as hypoglycaemic effects in rats in vivo, sage treatments9 have also shown effects on lipid metabolism in human cells in vitro. Lipids play many important roles in structural compartmentalisation and cellular signalling; for example it is worth noting that the steroid-hormone oestrogen – which is related to a traditional application of sage – is derived from lipids. Lipid metabolism has been broadly associated with ageing and longevity in many systems including C. elegans: The profile of lipids changes with age in the worm, and multiple genetic interventions have shown strong connections between elements of lipid metabolism (and sphingolipid metabolism in particular) and the core genetic pathways of ageing12-15. This includes Acid Sphingomyelinase-3 (asm-3)16,17, one of the genes up-regulated in response to both of the herbal treatments.

asm-3 is orthologous to the human gene Sphingomyelin Phosphodiesterase 1 (SMPD1), and orthologs of the human gene Glucocerebrosidase (GBA) are also present in the cluster of lipid metabolism genes up-regulated by the herbal treatments (Figure 2B). Misregulation of, or mutations in both of these genes have been associated with multiple diseases that cause neurodegenerative effects in humans, including Gaucher’s and Parkinson’s Disease in the case of GBA18,19 and Nieman-Pick Disease, dementia and Alzheimer’s Disease in the case of SMPD120-23. Thus, it is clear that correct homeostasis of sphingolipid metabolism – and most likely the bioactive lipid ceremide and its related metabolites in particular given their important roles including regulating cellular fate and autophagy – is important to cognitive health, and might provide another avenue for Sibelius&trade:Sage to confer anti-ageing cognitive benefits.

The mechanisms by which Sibelius™:Sage alters lipid metabolism via enzymes such as ASM and GBA, or has beneficial effects on obesity and metabolic health (with associated reduction in inflammation), and how these might contribute to benefits to cognitive health remain to be elucidated. However, investigating the molecular functions of herbal extracts in this way certainly opens the door to some fascinating questions as to the health benefits that herbal extracts can impart, which would likely remain hidden otherwise. Approaches such as a gene expression do not provide all of the answers, but they do help to direct our future research efforts at Sibelius and most importantly help us to refine what we believe the right questions to ask are.

Kieron D. Edwards
Scientific Director at Sibelius Ltd.

References

  1. Edwards, K. Using in vivo models to support development of nutraceuticals that promote healthy ageing. Nutraceuticals Now 19–20 (2017).
  2. Bansal, A., Zhu, L. J., Yen, K. & Tissenbaum, H. A. Uncoupling lifespan and healthspan in Caenorhabditis elegans longevity mutants. Proc. Natl. Acad. Sci. 112, E277–E286 (2015).
  3. Bellantuono, I. Find drugs that delay many diseases of old age. Nature 554, 293–295 (2018).
  4. Scholey, A. B. et al. An extract of Salvia (sage) with anticholinesterase properties improves memory and attention in healthy older volunteers. Psychopharmacology (Berl.) 198, 127–139 (2008).
  5. Hasselmo, M. E. The role of acetylcholine in learning and memory. Curr. Opin. Neurobiol. 16, 710–715 (2006).
  6. Cacabelos, R. Donepezil in Alzheimer’s disease: From conventional trials to pharmacogenetics. Neuropsychiatr. Dis. Treat. 3, 303–333 (2007).
  7. McDonnell, A. M. & Dang, C. yen H. Basic Review of the Cytochrome P450 System. J. Adv. Pract. Oncol. 4, 263–268 (2013).
  8. Lima, C. F., Azevedo, M. F., Araujo, R., Fernandes-Ferreira, M. & Pereira-Wilson, C. Metformin-like effect of Salvia of cinalis (common sage): is it useful in diabetes prevention? Br. J. Nutr. 96, 326 (2006).
  9. Khedher, M. R. B. et al. Preventive effects of salvia of cinalis leaf extract on insulin resistance and inflammation, in high fat diet- induced-obesity mice model. PeerJ Prepr. (2017).
  10. Dantzer, R., O’Connor, J. C., Freund, G. G., Johnson, R. W. & Kelley, K. W. From inflammation to sickness and depression: when the immune system subjugates the brain. Nat. Rev. Neurosci. 9, 46–56 (2008).
  11. Besser, L. M. et al. Body Mass Index, Weight Change, and Clinical Progression in Mild Cognitive Impairment and Alzheimer Disease: Alzheimer Dis. Assoc. Disord. 28, 36–43 (2014).
  12. Hou, N. S. & Taubert, S. Function and Regulation of Lipid Biology in Caenorhabditis elegans Aging. Front. Physiol. 3, (2012).
  13. Menuz, V. et al. Protection of C. elegans from Anoxia by HYL-2 Ceramide Synthase. Science 324, 381–384 (2009).
  14. Mosbech, M.-B. et al. Functional Loss of Two Synthases Ceramide Elicits Autophagy-Dependent Lifespan Extension in C. elegans. PLoS ONE 8, e70087 (2013).
  15. Huang, X., Liu, J. & Dickson, R. C. Down-regulating sphingolipid synthesis increases yeast lifespan. PLoS Genet. 8, e1002493 (2012).
  16. Kim, Y. & Sun, H. Functional genomic approach to identify novel genes involved in the regulation of oxidative stress resistance and animal lifespan. Aging Cell 6, 489–503 (2007).
  17. Kim, Y. & Sun, H. ASM-3 Acid Sphingomyelinase Functions as a Positive Regulator of the DAF-2/AGE-1 Signaling Pathway and Serves as a Novel Anti-Aging Target. PLoS ONE 7, e45890 (2012).
  18. Zhang, Y. et al. Effect of GBA Mutations on Phenotype of Parkinson’s Disease: A Study on Chinese Population and a Meta- Analysis. Park. Dis. 2015, 1–10 (2015).
  19. Dodge, J. C. Lipid Involvement in Neurodegenerative Diseases of the Motor System: Insights from Lysosomal Storage Diseases. Front. Mol. Neurosci. 10, (2017).
  20. Schuchman, E. H. Acid sphingomyelinase, cell membranes and human disease: Lessons from Niemann-Pick disease. FEBS Lett. 584, 1895–1900 (2010).
  21. Rhein, C. et al. Functional Implications of Novel Human Acid Sphingomyelinase Splice Variants. PLoS ONE 7, e35467 (2012).
  22. Lee, J. K., Jin, H. K. & Bae, J. ASM in Alzheimer’s disease. Oncotarget 6, 39389 (2015).
  23. Desnick, J. P. et al. Identification and characterization of eight novel SMPD1 mutations causing types A and B Niemann-Pick disease. Mol. Med. 16, 316 (2010).

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