Exploring the anti-aging potential of natural products and plant extracts in budding yeast Saccharomyces cerevisiae: A review

The Ras/Adenylate Cyclase/protein kinase A (PKA) Pathway

RAS genes encode small monomeric proteins called GTPases, which act as signal transducers in all eukaryotes. These Ras proteins are primarily involved in the modulating key cellular processes, including cell growth, proliferation, metabolism, and oncogenic transformation.^54,55 The yeast genes RAS1 and RAS2 are highly homologous to human ras proto-oncogenes, indicating the evolutionary conservation of the ras gene family. Genetic studies have showed that the RAS2 gene can be functionally interchangeable between yeast and humans.⁵⁶ Mutations in RAS genes have been shown to improve the oxidative stress resistance and increase survival rates in different model systems including yeast, C. elegans, drosophila, and mammalian cell lines (e.g., PC12 cells).⁵⁷ Chronological aging experiments first identified and characterized that the survival of yeast ras2Δ mutant increase by cent percent compared to wild-type yeast. Additionally, ras2Δ mutant cells demonstrated augmented resistance to both heat stress and oxidative stress.⁵⁷

In yeast, Ras 1/2 can directly activate adenylate cyclase (Cyr1), which is encoded by CYR1 gene (Figure 2). In addition to direct activation by Ras 1or 2, the Cyr1 can also be activated by the G protein-coupled receptor Gpr1.⁵⁸ These two activation ways for Cyr1 are induced when glucose is present in the growth medium.⁵⁹ Activated Cyr1, in turn, activates a cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA). In its inactive form, PKA exists as a tetramer of two regulatory subunits (Bcy1) and two catalytic subunits (Tpk1, Tpk2 or Tpk3) (Figure 2). Although the yeast PKA belongs to a different family of serine/threonine kinase, its two catalytic subunits are 35 to 42% identical to C. elegans and human AKT-1/AKT-2.⁶⁰

The binding of cAMP to the regulatory subunits leads to their dissociation from the complex and activation of the catalytic subunits.⁶¹ The activation of PKA is essential for cell growth and proliferation. When glucose is abundant, the Ras/cAMP/PKA pathway gets activated to induce the massive expression of genes that promote growth rate in yeast.⁶² The activated PKA inhibits Rim15, a PAS-kinase that integrates signals from different nutrient sensing signaling pathways such as TORC1, Sch9, PKA, and Pho80-Pho85 to transcription factors Msn2/4 and Gis1 (Figure 2).⁵³ The inhibition of Rim15 phosphorylation positively regulates the transcription factors such as Msn2/4 and Gis1. It is assumed that Rim15 also play a role in the regulation of genes involved in the glucose repression and cell cycle arrest.⁵³ In yeast, the transcription factors Msn2/4 and Gis1 bind respectively to the STRE (STress Responsive Element) and the PDS (post-diauxic shift) sequences, thereby activate stress resistance genes including SOD1 SOD2, CTT1, HSPs, and DDR2 (Figure 2). In contrast, the Ras/cAMP/PKA pathway inactivation leads to enhanced heat stress resistance, by partly activating Msn2 and Msn4, which induce the expression of genes encoding for several heat shock proteins (HSPs), superoxide dismutases (SOD1 and SOD2) catalase (CTT1), and the DNA damage inducible gene DDR2.^60,63

While the critical functioning of Msn2 and Msn4 are vital for the extension of CLs in both ras2 and cyr1 mutants, previous studies have also demonstrated the essential role of another transcription factor, Gis1, in enhancing stress resistance and extending CLS under Ras/PKA inactivation.⁶⁴ Thus, the critical roles of Ras/Adenylate Cyclase/PKA Pathway in the regulation of metabolism, stress resistance, proliferation, and longevity in S. cerevisiae are mainly linked to the availability of nutrients.⁶³

It was also reported that mutations in CYR1 gene, as well as mutants with impaired synthesis of cAMP, cause extension of CLS in yeast,⁶⁰ indicating the potential involvement of PKA as a pro-aging pathway. Research evidences also suggests that the pro-aging role of the Ras/adenylate cyclase/PKA pathway seems to be conserved from yeast to mammals. Studies in mice deficient in p66Shc, a signal transducer that might trigger cell proliferation via Ras activation, demonstrated increased oxidative stress resistance and lived 30% longer than wild type.⁶⁵ Yan et al. further strengthened the critical role of adenylate cyclase (AC5) in the regulation of mammalian aging. They reported that both myocytes and fibroblasts isolated from AC5 knock out mice showed enhanced oxidative stress resistance compared to control cells. Additionally, tissues from AC5 knock out mice, such as brain, heart, and kidneys, showed elevated levels of MnSOD and these mice lived 30% longer than their control littermates, implying the fundamental role of adenylate cyclase AC5 in regulating mammalian lifespan and oxidative stress resistance.⁶⁶

Enns et al. (2009) provided evidence for the highly conserved pro-aging role of PKA in mammals though experimental studies in RIIB null mice. In mammals, the cAMP dependent-protein kinase A is composed of two regulatory (RI and RII) subunits and two catalytic (C) subunits. The RI and RII subunits exist as two isoforms called RIα, RIβ, RIIα, and RIIβ.⁶⁷ The RIIβ subunit is predominantly expressed in brown and white adipose tissue and in brain, tissues involved in the regulation of energy homeostasis. RIIβ null mice showed lean body mass and increased median and maximum lifespan compared to wild type littermates. This further supports the essential role of the PKA pathway in mammalian aging as well.⁶⁸

The TOR-Sch9 pathway

In response to nutrients, particularly, amino acids, the target of rapamycin (TOR) pathway stimulates anabolic processes and represses catabolic processes such as autophagy, to promote cell growth.⁶⁹ Target of rapamycin (TOR), a serine threonine kinase was first identified in the budding yeast S. cerevisiae.⁷⁰ S. cerevisiae has two TOR protein kinases, Tor1 and Tor2. TOR forms two structurally and functionally distinct complexes: Tor1 containing TOR complex 1 (TORC1) and Tor2 containing TOR complex 1 (TORC2).⁷¹ While TORC1 responds to amino acids, controlling cell growth, protein synthesis, metabolism, autophagy, and aging, TORC2 is implicated in the sphingolipid biogenesis, endocytosis, and actin organization. TORC1 is highly conserved between yeast and mammals.^72,73 It consists of subunits such as, TOR1 or TOR2 (ortholog of mammalian TOR, mTOR), Kog1(ortholog of RAPTOR, regulatory-associated protein of TOR), and Lst8 (ortholog of mLST8, mammalian lethal with SEC13 protein 8). Only TORC1 is sensitive to the drugs rapamycin, but not TORC2.⁷⁴

The TORC1 pathway is the second major pathway implicated in the regulation of aging in yeast (Figure 2). In response to amino acids levels, Rag GTPases (members of the Ras superfamily) induce activation of mTORC1 signaling in mammals.⁷⁴ The yeast Gtr1p and Gtr2p are orthologous to mammalian Rag A/B and Rag C/D GTPases, respectively. The amino acid induced activation of TORC1 in yeast is mediated by Gtr1p-GTP and Gtr2p-GDP heterodimer, which interacts with Ego1 (or Gse2) (an ortholog of the mammalian protein MP1), and Ego3 (or Gse1) (an ortholog of the mammalian protein p14), and forms an EGO/GSE complex (Figure 2). This EGOC is localized to the vacuolar membrane via Ego1 subunit. The GTP bound Gtr1p of EGO/GSE complex activates TORC1, which in turn activates its downstream Sch9 protein kinase (a homolog of human ribosomal S6 kinase 1, S6K1) by phosphorylation.⁷⁵

S. cerevisiae consists of two functionally redundant protein kinases Pkh1 and Pkh2 (homologs of mammalian PDPK1, phosphoinositide-dependent protein kinase-1), which are involved in maintaining the cell wall.⁷⁶ Both Pkh 1 and 2 protein kinases can also phosphorylate Sch9 kinase.⁷⁷ In yeast, when glucose levels are low, another protein kinase called SNF1 (sucrose non-fermenting 1 kinase, a homolog of human AMPK) becomes activated and phosphorylate Sch9 (Figure 2), promoting yeast adaptation to glucose depletion. Thus, the yeast AMPK, SNF1 acts as a key regulator of energy homeostasis.⁷⁸

Activation of Tor and Sch9 lead to inactivation of Rim15 kinase and the stress resistance transcription factor Gis1 (Figure 2), both of which are required for maximum CLS extension.⁴³ Similar to Msn2 and Msn4, Gis1 induces the expression of mitochondrial superoxide dismutase enzyme (MnSOD), which is required for the effect of sch9 deletion on CLS. The deletion of SCH9 can extend both RLS and CLS of yeast.^60,79,80 Additionally, the combined deletion of SCH9 along with RAS2 can furthermore extend the yeast CLS compared to SCH9 deletion alone.⁸⁰ Both CLS and RLS can be extended by the loss or inhibition of TOR1 via inactivating the downstream Sch9.⁵² Aging yeast is characterized by the elevated levels of ROS. In contrast, reduced levels of ROS were observed in the long-lived mutants deficient in Ras-AC-PKA or Tor-Sch9 signaling.³⁴

During CLS, glucose levels decrease gradually and accumulation of non-fermentable carbon sources such as ethanol and acetic acid takes place. Both ethanol and acetic acid promote the chronological aging in yeast.⁴⁵ Loss of either TOR1or SCH9 slows down the aging process partly by lowering respiration and stimulating the exhaustion of ethanol and acetic acid and the elevating the levels of glycerol in the nutrient medium.⁸¹ Since S. cerevisiae cannot ferment either acetic acid or ethanol, their depletion by mutations in TOR or SCH9 can prolong the CLS by creating similar conditions usually caused by dietary restriction. It is important to note that the yeast cells deficient in TOR-Sch9 or Ras-AC-PKA signaling pathways exhibit prolonged CLS during incubation in water or in a medium which is either deficient or composed of low amounts of acetic acid and ethanol.^53,82

Natural products/plant extracts vs. CLS

Several studies indicate that various natural products and plant extracts have the potential to extend CLS in yeast. These natural products belong to different phytochemical classes, such as: flavonoids (e.g., quercetin), carotenoids (e.g., astaxanthin),^85,86 sesquiterpenes (e.g., artesunate),⁸⁷ triterpenoids (e.g., betulinic acid),⁸⁸ flavanones (e.g., Hesperitin)⁸⁹ and flavonoid glycosides (e.g., Neohesperidin),⁸⁹ polyphenols (e.g., chlorogenic acid),⁹⁰ saponins (e.g., Ginsengoside Rg1),⁹¹ lignans (e.g., Magnolol),⁹² etc. Additionally, crude extracts prepared from different plant species were shown to delay yeast chronological aging. In the following section, we summarize the effects of some natural products and plant extracts on the chronological aging in yeast S. cerevisiae. Table 1 lists the natural compounds/plant extracts, their concentrations, phytochemical class, type of growth medium used for CLS experiments, and their anti-aging mechanisms in different S. cerevisiae strain backgrounds.

Acetyl L-carnitine

Acetyl-L-carnitine (ALC) is an endogenous molecule synthesized in the body from L-carnitine. ALC plays a key role in the energy metabolism and it has been reported to exhibit a plethora of pharmacological properties, including neuroprotective activities. It has also been used as a dietary supplement due to its potent therapeutic effects. Researchers have shown the anti-apoptotic and anti-aging ability of ALC in yeast model. ALC was found to inhibit mitochondrial fission and subsequently improve mitochondrial functioning. Yeast gene MCA1 encodes a Ca+2-dependent protease which plays a key role in the regulation of apoptosis. It was found that the mitoprotective effects of ALC were mediated through the yeast metacaspase (Yca1) and thus to its anti-apoptotic activity. ALC was also shown to extend the CLS of yeast cells.⁹³

Astaxanthin

Astaxanthin is a carotenoid compound with superior antioxidant ability than many other natural antioxidant molecules. Sudarshan et al. conducted a study to test the antioxidant, anti-apoptotic and anti-aging properties of astaxanthin using yeast model. Astaxanthin treatment prevented oxidative stress-induced surge in MDA and ROS levels and reduction in superoxide dismutase and glutathione levels, thereby increasing the percentage viability of antioxidant gene-deleted yeast mutants by 20-40%. In addition, astaxanthin also prevented the apoptosis of aged cells and subsequently increased the viability of yeast cells during the CLS. Astaxanthin’s antioxidant and anti-apoptotic effects are the possible reason for its anti-aging activity.⁸⁵ It was also suggested that anti-apoptotic effects of astaxanthin are also due to its ability to prevent nuclear fragmentation and chromatin condensation in yeast cells.⁸⁶ Sudarshan et al. also found that astaxanthin improved oxidative stress resistance and increased the viability of yeast DNA damage repair gene-deleted mutant cells. Astaxanthin was shown to prevent the accumulation of endogenous DNA damage marker (8-hydroxy-2-deoxyguanosine) levels in yeast cells. The anti-aging effects of astaxanthin were also suggested to be due to its ability to inhibit the accumulation of mutation during chronological aging of DNA damage repair gene-deleted mutant yeast cells.⁸⁶

Artesunate

Artesunate is a semi-synthetic derivative of the antimalarial drug artemisinin. Previous studies have shown that artesunate exerts anti-aging effects similar to caloric restriction, indicating the CR mimetic effects of artesunate. From the whole-transcriptome profile analysis studies, it was revealed that artesunate mimics CR-triggered nitric oxide to induce antioxidant defense systems, thereby preventing ROS accumulation and mitigating oxidative stress, subsequently extending yeast lifespan.⁸⁷

Betulinic acid

Betulinic acid (BA) is a pentacyclic triterpenoid present in some plant species. A recent study reported that betulinic acid extends the CLS of yeast cells by mitigating oxidative stress-induced apoptosis. The anti-aging effects of betulinic acid were found to be mediated by the induction of genes associated with heat shock stress response and autophagy.⁸⁸

Citrus flavonoids

Flavonoids have been reported to exert a plethora of pharmacological activities including antioxidant, anti-inflammatory, anti-cancer, anti-neurodegenerative, anti-aging, among others. Researchers have investigated the anti-aging effects of citrus flavonoids including naringin, hesperedin, hesperitin, and neohesperidin on the chronological aging of yeast. Among the tested flavonoids, neohesperidin significantly prevented accumulation of ROS and extended the chronological life span of yeast in a concentration-dependent manner.⁸⁹ In another study, treatment with hesperidin significantly induced the expression levels of SOD and sirtuin2 (SIR2) and prevented ROS accumulation in yeast. In yeast, UTH1 gene encodes Uth1p, which is activated by oxidative stress, senescence, and TOR-dependent autophagy; all these events subsequently lead to cell death in yeast. Pretreatment with hesperidin, but not its aglycon hesperetin, significantly inhibited the expression of UTH1 gene, thereby extending yeast lifespan.⁹⁴

4-N-furfurylcytosine

Previous studies have demonstrated that purine and pyrimidine derivates can exhibit promising health promoting effects.^95,96 Pawelczak et al. investigated the antiaging effects of 4-N-Furfurylcytosine (FC), a cytosine derivative using model S. cerevisiae. They found that treatment with FC increased the percentage viability of yeast cells during CLS in a concentration-dependent manner. In addition, treatment with FC boosted the mitochondrial activity and reduced intracellular levels of ROS. It was also demonstrated that FC could limit TORC1 signaling in yeast. This points to the fact that FC’s anti-aging activity is through the inhibition of TOR signaling pathway.⁹⁷

Galactan exopolysaccharide

A recent study using yeast models investigated the antioxidant and anti-aging properties of galactan exopolysaccharide isolated from a gram-positive Weissella confusa. Galactan exopolysaccharide was shown to prevent the oxidative stress induced rises in ROS levels and enhance the viability of yeast cells exposed to hydrogen peroxide as an oxidant used in this study. In addition, galactan exopolysaccharide treatment significantly increased the viability of both wild type and antioxidant gene-deleted mutant (sod2∆). The study suggested that the antioxidant potential of the galactan exopolysaccharide could be the possible reason for its anti-aging activity.⁹⁸

Ginsengosides

Ginsenosides are major pharmacological compounds that are unique to the plant species Panax ginseng C. A. Meyer (ginseng). Ginsenosides have been demonstrated to extend the life span of different model organism. In a recent study, researchers treated yeast cells with ginsenoside Rg1 and found an augmented antioxidant stress response and concomitant reduction in ROS levels and apoptosis, thereby increasing the viability during aging in yeast model. It was also demonstrated that ginsenoside Rg1 treatment resulted in increased mitochondrial bioenergetics and glycolytic enzymes, thereby improving metabolic homeostasis and delaying aging in S. cerevisiae.⁹¹

Glutamic acid and methionine

A study⁴³ reported that the composition of amino acids in aging media can affect the chronological life span of yeast cells. The presence of non-essential amino acids methionine and glutamic acid have been reported to greatly influence the survival rate of yeast cells during aging. Precisely, low levels of methionine and high levels of glutamic acid in the yeast nutrient media led to extended lifespan in yeast model. In contrast, increasing levels of methionine and reducing levels of glutamic acid caused a decrease in yeast life span. Therefore, it can be concluded that amino acid composition is a critical factor for controlling yeast aging.⁹⁹

Lithocholic acid

Previous studies have reported that lithocholic acid, a bile acid, can increase yeast cell survival during chronological aging.^100–103 Interestingly, lithocholioc acid was also shown to significantly enhance the survival rate of yeast especially under caloric restriction conditions. Lithocholic acid treatment was shown to modulate several cellular pathways, including carbohydrate and lipid metabolism, mitochondrial structure and function, liponecrotic and apoptotic cell death of yeast during chronological aging.¹⁰⁰ It is important to note that lithocholic acid’s anti-aging effects under caloric restriction are found to be prominent only when this compound is added to the growth medium either at logarithmic/diauxic and early stationary stages of yeast aging. Addition of lithocholic acid either at logarithmic/diauxic and early stationary stages induced the activation of several longevity related cellular processes which ultimately triggering the enhanced yeast cell survival during aging.¹⁰¹ Beach et al. also demonstrated that lithocholic acid exerts its anti-aging effects through the modulation of the expression of different transcription factors including Aft1p, Hog1p, Msn2/4p, Rtg1p- Rtg3p, Sfp1p, Skn7p, and Yap1p. Each of these transcription factors in turn alter the levels of several intra and extra mitochondrial proteome, and subsequent maintenance of mitochondrial function. Altogether, Beach et al. suggested that lithocholic acid’ anti-aging effects are mainly through the modulation of aging-related transcriptional landscape.¹⁰² Also, it was discovered that lithocholic bile acid accumulates in mitochondria, and alters the mitochondrial membrane lipidome which is crucial for the restoration of mitochondrial proteome, subsequently an improved mitochondrial function and increased chronological aging by lithocholic acid.¹⁰³

Magnolol

In our previous study, we reported the anti-aging effects of magnolol, a natural polyphenol. Magnolol enhanced the stress resistance of yeast cells exposed to hydrogen peroxide, an oxidizing agent. In addition, magnolol increased the viability of the short-lived yeast mutant sod1∆, which lacks the antioxidant enzyme superoxide dismutase. Our study suggested that the anti-aging effects of magnolol occur mainly through the modulation of oxidative stress during yeast chronological aging.⁹²

Morusin and mulberrin

Mulberry leaves are rich in flavonoids such as morusin and mulberrin, and these have been demonstrated to enhance the survival rate during yeast chronological aging. It was also found that morusin and mulberrin exert their anti-aging effects via targeting the SCH9, a major target of TORC1 in budding yeast S. cerevisiae.¹⁰⁴ Budding yeast Sch9 is the major substrate of the TORC1. Yeast Sch9 functions analogously to S6K1, the mammalian TORC1 substrate. Yeast TORC1 phosphorylates about six amino acid residues of Sch9, indicating that the TORC1-dependent phosphorylation of Sch9 is essential for its activity.¹⁰⁵ Activated Sch9 plays a key role in the regulation of several cellular processes including protein synthesis (via modulating ribosome synthesis and translation initiation), cell cycle, and aging. Deletion of SCH9 causes growth defects such as reduction in cell size and growth rate; however, deletion of SCH9 leads to enhanced temperature tolerance as well chronological and replica life spans in budding yeast. The longevity-extending effects due to SCH9 deletion are explained by the augmented oxidative stress response and reduction in age-associated mutation rate.¹⁰⁶

Myricetin

In another study, using budding yeast as a model, researchers investigated antioxidant and anti-aging effects of myricetin, a polyphenolic flavonoid compound which is composed of a pyrogallol ring in the B ring of its structure. Myricetin’s antioxidant effects are attributed to the presence of a hydroxyl group in its B ring. Treatment with myricetin reduced the levels of ROS and protein carbonyl content, thereby enhancing the oxidative stress resistance of yeast cells exposed to hydrogen peroxide. Myricetin was found to inhibit H₂O₂-induced glutathione oxidation, but did not enhance endogenous antioxidant enzymatic activity in yeast. Additionally, myricetin treatment significantly prevented age associated oxidative stress and extended the CLS of yeast mutant lacking mitochondrial superoxide dis-mutase (sod2Δ).¹⁰⁷

Quercetin

It is noteworthy that several of the yeast genes, such as, PEP4 and TEL1, share homology with human genes of diseases relevance, such as CTSD and ATM, respectively.^108,109 In a previous study, we demonstrated the protective effects of quercetin on S. cerevisiae pep4Δ mutant that lack the PEP4 gene encoding a vacuolar endopeptidase proteinase A. This vacuolar proteinase A is a homolog of the human cathepsin D (encoded by CTSD), which plays a key role in the normal development and maintenance of neurons in the central nervous system. Particularly, it is essential for the degradation of proteins linked to several neurodegenerative diseases such as Parkinson’s disease, Huntington disease, neuronal ceroid lipofuscinosis, and Alzheimer’s disease.¹⁰⁸ Yeast pep4Δ cells are found to be highly sensitive to oxidative and apoptotic stressors. However, in our study, we demonstrated that quercetin treatment reduced ROS levels and apoptotic markers, thereby increasing the percentage viability of yeast pep4Δ cells during chronological aging.¹⁰⁸

The mammalian serine/threonine protein kinase ATM plays an important role in DNA damage sensitivity, cell cycle checkpoint deficiency, cancer incidence and telomere length maintenance. Mutation in ATM gene is linked to elevated oxidative damage, premature aging, and apoptosis. In another study, we also investigated the protective effects of quercetin on the sensitivity of S. cerevisiae tel1Δ cells lacking Tel1p, which is a homolog of the human ATM (Ataxia Telangiectasia Mutated) gene mutation. Our study results showed that quercetin treatment prevented ROS accumulation and thereby enhanced the stress resistance of tel1Δ cells exposed to a variety of oxidizing agents. Furthermore, treatment with quercetin prevented apoptotic death of yeast tel1Δ cells and increased cell viability during chronological aging.¹⁰⁹

Spermidine

Spermidine has been reported to inhibit histone acetyltransferases and subsequent deacetylation of histone H3, thereby preventing oxidative stress and necrosis in yeast aging. On the contrary, reduction of endogenous polyamines caused hyperacetylation, accumulation of ROS, necrotic cell death and a diminished life span. It is important to note that treatment with spermidine causes alteration in acetylation status of the chromatin, resulting in a substantial increase in the autophagy in different model organisms, including yeast. This suggests that spermidine’s anti-aging effects are mediated through its ability to stimulate the autophagy pathway.¹¹⁰

Tanshinones

Wu et al. found that the dried roots of Salvia miltiorrhiza Bunge have substantial longevity extending effects. They reported that tanshinones (e.g., cryptotanshinone, tanshinone I, and tanshinone IIa) are pharmacologically active components present in the roots of S. miltiorrhiza. Precisely, cryptotanshinone has been reported to extend the CLS of yeast wild type as well as yeast mutant lacking mitochondrial superoxide dismutase (sod2Δ). Their study suggests that the cryptotanshinone-induced anti-aging effects are modulated through the involvement of nutrient-sensing protein kinases such as Tor1, Sch9, and Gcn2.¹¹¹

Almond extracts

Almond (Prunus dulcis (Mill.) D.A. Webb) is a major nut crop worldwide. Multiple health benefits associated with the consumption of almonds are due to the phenolic-rich almond skin. Previous studies have found that pre-treatment with almond skin extract and chlorogenic acid significantly extended the lifespan of yeast. Both almond extract and chlorogenic acid improved the mitochondrial function during chronological aging of yeast cells by reducing the accumulation of free radicals (including ROS and RNS) and preserving mitochondrial membrane potential (MMP). Furthermore, treatment with almond extract and chlorogenic acid augmented the oxidative stress response by inducing the expression levels of SIR2 and SOD1 genes and decreasing the endogenous levels of lipid peroxides, protein carbonyls and 8-hydroxy-2-deoxyguanosine (8-OHdG) in yeast cells. Altogether, it can be suggested that the longevity-extending effects of almond extract are ascribed to its ability to ameliorate oxidative stress in yeast cells.⁹⁰

Coffee

Czachor, J., et al. demonstrated the lifespan-extending effects of coffee infusions in S. cerevisiae. Coffee, especially the Coffea robusta type, was found to exhibit superior antioxidant effects than the Coffea arabica type, thereby protecting cells from ROS-induced DNA damage and additionally improving the metabolic activity in yeast cells. overall, it can be suggested that coffee exhibits health benefits mainly through the reduction of ROS accumulation and the enhancement of metabolic activity.¹¹²

Polyalthia longifolia

Polyalthia longifolia is a polyphenol-rich traditional medicinal plant that has long been used for its rejuvenation capacity. P. longifolia has been reported to exhibit potent pharmacological activities including antioxidant and hepatoprotective activities. A previous study,⁶³ tested the anti-aging activity of methanolic leaf extract of P. longifolia using yeast CLS model and found that the methanolic leaf extract enhanced the viability of yeast cells and extended the CLS of yeast. It was reported that methanolic leaf extract significantly prevented the accumulation of H₂O₂-induced ROS levels and increased GSH levels. Furthermore, treatment with methanolic leaf extract strikingly induced the expression levels of SOD and SIRT1 genes. Overall, it can be suggested that P. longifolia exerts its anti-aging effects through the modulation of oxidative stress response and SIRT1 gene.¹¹³ It was also reported that methanolic leaf extract of P. longifolia extended the RLS of yeast via ameliorating the apoptotic features of replicatively ageing yeast cells.¹¹⁴

Rice bran extract

Pigmented rice is the functional food in many countries including India, China, and Japan, and it is the richest source of polyphenols.¹¹⁵ It has been reported that red rice bran extract can prevent the accumulation of ROS levels, maintain plasma membrane integrity and extend the CLS of yeast cells. The anti-aging mechanism of action of rice bran extract is through the modulation of the TOR1 and SIR2-dependent pathways.¹¹⁶

Sacred lotus stamen extract

The stamen of lotus (Nelumbo nucifera) is rich in flavonoids and has long been widely used in Indian traditional medicine. Tungmunnithum et al. reported that treatment with lotus stamen extract significantly enhanced the antioxidant status (both enzymatic activity and gene expression levels of SOD1 and SIRT2) and extended the CLS of yeast cells. Interestingly, the longevity extending effects of lotus stamen extract were reported to be superior to the resveratrol treatment.¹¹⁷

Previous studies also demonstrated that a variety of plant extracts can extend the CLS in yeast by modulating both pro-aging and anti-aging protein kinases. Plants extracts of Apium graveolens and Cimicifuga racemosa have been shown to stimulate the anti-aging protein kinases Rim15 and SNF1, respectively.¹¹⁸ Both Valeriana officinalis and Ginkgo biloba extracts were shown to exert inhibitory action on the pro-aging PKA pathway, whereas, the anti-chronological aging effects of Cimicifuga racemosa and Salix alba were shown to be modulated via inhibitory action on the pro-aging kinases Sch9 and TORC1, respectively.¹¹⁸

The anti-aging potential of these plant extracts were also attributed to their ability to enhance hormetic stress response in yeast. Precisely, these extracts were shown to increase mitochondrial respiration and membrane potential, decrease or increase ROS, enhance resistance to both thermal and oxidative stress, and mitigate oxidative damage to protein, lipids, and DNA.^119,120 Additionally, Salix alba extract was demonstrated to exhibit anti-chronological aging effects via remodeling of both intracellular and mitochondrial lipid metabolism, particularly, by decreasing intracellular free fatty acid levels, resulting in delay in the age-related liponecrotic yeast cell death.¹²¹ Kwong, M.M.Y. et al. (2021) screened for the anti-aging potential of 222 plant extracts and their findings revealed that two plant extracts namely Manihot esculenta and Wodyetia bifurcata could extend the CLS via inducing the oxidative stress response pathways in yeast.¹²²

Natural products/plant extracts vs. RLS

Researcher also demonstrated the potential of natural products and plant extracts to extend the RLS in yeast. These natural products belong to different phytochemical classes, such as secoiridoid glycoside (e.g., amarogentin),¹²⁵ flavonone glycoside (e.g., hesperidin),⁹⁴ triterpenoid glycoside (e.g., cucurbitane glycoside),¹²⁶ ergosterol derivatives (e.g. ganodermasides A and B),¹²⁷ phospholipid (e.g., lysophospholipid),¹²⁸ flavonoid (e.g., phloridzin),¹²⁹ stilbenoid (e.g., reservatrol),¹³⁰ and polyphenolic glycosides (e.g., parishin).¹³¹ Others also demonstrated that crude extracts prepared from different plant species including Polyalthia longifolia, ¹¹⁴ Psoralea corylifolia, ¹³² and Pterocarpus marsupium¹³³ extended RLS in yeast. In the following section, we summarize the effects of some natural products and plant extracts on the replicative aging in yeast S. cerevisiae. Table 2 lists the natural compounds/plant extracts, their concentrations, phytochemical class, type of growth medium used for RLS experiments, and their anti-aging mechanisms in different S. cerevisiae strain backgrounds.

Amarogentin

Disasa et al. isolated a secoiridoid glycoside amarogentin from a Chinese traditional medicinal plant, Gentiana rigescens Franch. Amarogentin was reported to enhance both the enzymatic activities and expression levels of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx), thereby improving the viability of yeast cells exposed to oxidative stress. Furthermore, treatment with amarogentin resulted in an enhanced RLS of yeast wild type; however, this compound was not shown to extend the lifespan of yeast mutants sod1Δ, sod2Δ, uthΔ, and skn7Δ, suggesting that the anti-aging effects of amarogentin are mainly through the regulation of antioxidative stress as well as by the regulation of UTH1, SKN7, SOD1, and yeast SOD2 gene expression.¹²⁵

Cucurbitacins

Cucurbitacins are a class of tetracyclic triterpenoids that are abundant in plants belonging to the family Cucurbitaceae. Cucurbitacins have been reported to exhibit strong anticancer activity, and are divided into 12 classes from A to T with over 200 derivatives.¹³⁴ Cucurbitacin B is the most abundant and active member of the cucurbitacins.¹³⁵ Researchers have discovered that cucurbitacin B can extend both replicative and chronological life spans in yeast. Treatment with cucurbitacin B enhanced the lifespan of yeast by promoting the expression of ATG32. Cucurbitacin B could not increase the lifespan of yeast mutants devoid of autophagy related genes (ATG2 and ATG32), pointing to the fact that the anti-aging effects of cucurbitacin B are mainly through the induction of autophagy in yeast.

It was also demonstrated that cucurbitacin B can ameliorate oxidative stress levels mainly through the induction of superoxidase dismutase activity (via enhanced expression of SOD1 and SOD2) and the prevention of accumulation of oxidative stress markers including ROS and malondialdehyde (MDA). Furthermore, researchers also showed that the anti-aging effects of cucurbitacin B are also mediated through the regulation of expression of other aging-related genes such as UTH1 as well as SKN7. Cucurbitacin B’s anti-aging effects are attributed to its ability to ameliorate oxidative stress and regulate autophagy and age-related genes in yeast.¹³⁶

Additionally, cucurbitane glycoside isolated from the methanol extracts of Momordica charantia L. fruits has been reported to significantly extend the RLS of K6001 budding yeast mainly through suppressing ROS levels and the oxidative stress burden. Treatment with cucurbitane glycoside resulted in a decrease in the expression levels of UTH1 and SKN7 and an increase in the expression levels of SOD1 and SOD2. However, cucurbitane glycoside was not able to extend the RLS of the yeast mutants uth1Δ, skn7Δ, sod1Δ, and sod2Δ, suggesting that cucurbitane glycoside exerts antiaging effects via antioxidative stress and regulation of yeast UTH1, SKN7, SOD1, and SOD2 gene expression.¹²⁶

Copper and iron supplementation

Previous studies were conducted to demonstrate the effects of supplementation of copper and iron on the yeast RLS. It was found that addition of copper to the growth media increased RLS via the activation of multicopper oxidase enzyme (Fet3p) that works in combination with another enzyme called iron permease (Ftr1p), allowing for high intake of iron into yeast cells. Furthermore, it is important to note that high levels of iron-mediated anti-aging effects are dependent on the multicopper oxidase enzyme. However, the life span-extending effects of both copper and iron occurred in the growth medium supplemented with only glycerol as a carbon source but not glucose.

It was also demonstrated that supplementation of either copper or iron prevented the accumulation of ROS levels, thereby enhancing the replicative life span of yeast mutants lacking antioxidant enzymes.^137,138 The iron-mediated anti-aging effects occur mainly through the enhanced expression of genes related to the mitochondrial metabolism especially tricarboxylic acid (TCA) cycle and electron transport chain, subsequently elevating levels of adenosine triphosphate (ATP), which is essential for increased survival cells during aging. Interestingly, supplementation of iron could also enhance the life span of yeast Snf1Δ mutant which lacks SNF1/AMPK protein kinase. Therefore, it can be suggested that the iron’s life span-extending effects occur via amelioration of mitochondrial energy metabolism in yeast.¹³⁹

Ganodermasides A and B

Medicinal mushrooms have been reported to exhibit anti-aging activity. In a study,³⁸ researchers isolated two ergosterol derivatives, namely ganodermasides A and B, from the spores of the medicinal mushroom Ganoderma lucidum. They showed that both ganodermasides A and B could extend the replicative life span of S. cerevisiae through the modulation of the expression of UTH. In yeast, different transcription factors such as Skn7, Yap1, and Mot3 play a crucial role in oxidative stress resistance. It was suggested that upon phosphorylation, these transcription factors get activated, thereby regulating the expression of UTH1 gene by binding to its upstream promoter region. It was reported that polyphenols’ anti-aging activity occurs through the activation of Skn7 and subsequent increased expression of the UTH1 gene.¹²⁷

Lysophosphatidic acid

In another study, Sun Y et al., isolated lysophosphatidic acid (LA) from the seeds of Arabidopsis thaliana and showed that LA augmented oxidative stress resistance, thereby promoting the extension of RLS in yeast. However, LA was not able to extend the replicative lifespan of mutants uth1Δ, skn7Δ, sod1Δ, and sod2Δ. This study suggests that LA’s anti-aging effects are possibly through the amelioration of antioxidant status of yeast cells and the genes of UTH1, SKN7, and SOD may also be involved in the action.¹²⁸

Nicotinamide adenine dinucleotide (NAD+) precursors

Alterations in the biosynthesis and regulation of nicotinamide adenine dinucleotide (NAD+) plays a crucial role in the progression of aging as well as pathophysiology of several age-related chronic diseases.¹⁴⁰ NAD + plays a crucial role in several metabolic processes in the cells. Most importantly, NAD+ functions as a co-factor in redox reactions and it is reduced to NADH in many metabolic pathways including glycolysis, the citric acid cycle, and fatty acid metabolism. Adequate intracellular levels of NAD+ are maintained through the combined action of NAD(+) biosynthesis and salvage pathways.

Several key cellular enzymes including sirtuin protein deacetylases and poly-ADP-ribose polymerases (PARPs) are NAD+ dependent, and use NAD+ as a substrate in many cellular processes, including aging. Aging is associated with a reduction in NAD+ levels, leading to alterations in several metabolic processes that are NAD+-dependent, which in turn leads to the ageing-associated physiological/functional decline. Therefore, supplementation with diets rich in three important NAD+ precursors 1) nicotinamide, 2) nicotinic acid (e.g., protein rich foods such as cereals, peanuts, meat, fish) and 3) nicotinamide riboside (e.g., milk, cabbage, cucumber) ameliorate age-related decline in NAD+ levels and help in the extension of active longevity.¹⁴¹

Nicotinamide riboside is a natural product present in milk.³⁰ NAD+ is also required for the proper functioning of Sir2 and extension of RLS in budding yeast. Alterations in NAD+ levels negatively affect the RLS of yeast. In eukaryotes, nicotinamide riboside kinases (Nrk1 and Nrk2) catalyze the phosphorylation of nicotinamide riboside to nicotinamide mononucleotide, the precursor of NAD+. A previous study reported that the supplementation of nicotinamide riboside increased the levels of NAD+ via activation of both Nrk1-dependent pathway and the Urh1/Pnp1/Meu1 pathway (an Nrk1-independent pathway).

Increased NAD+ levels in turn lead to enhanced Sir2-dependent gene silencing, thereby extending RLS in yeast.¹⁴² The Sir2 enzyme deacetylates the lysine residues of histones, thereby silence all heterochromatin-like regions including telomeres, rDNA, and the hidden mating type loci HML/HMR (Hidden MAT Left/ (Hidden MAT Right).¹⁴³ In the S. cerevisiae NAD+ salvage pathway, nicotinamide is recycled from NAD+ by the Sir2 mediated deacetylation reaction.

The nicotinamidase (Pnc1) catalyzes the conversion of nicotinamide to nicotinic acid, which is further converted to nicotinamide mononucleotide by the enzyme nicotinamide phosphoribosyltransferase (Npt1). Isonicotinamide (a compound similar in shape and electrical properties to nicotinamide) was shown to elevate intracellular levels of NAD+ through the yeast NAD+ salvage pathway, leading to an increase in Sir2 activation. This, in turn, enahnce normal silencing at the rDNA locus in yeast and extension of yeast RLS.¹⁴⁴

Parishin

Parishin is a phenolic glucoside isolated from Gastrodia elata, a Chinese traditional medicinal plant. Treatment with parishin significantly increased cell viability under oxidative stress and extended the replicative life span of K6001 yeast. Parishin treatment significantly induced the expression of levels of SIR2 and SOD activity, while preventing the accumulation of ROS and lipid peroxidation. However, Parishin did not increase the RLS of sod1Δ, sod2Δ, uth1Δ, and skn7Δ mutants of K6001 yeast.

Treatment with parishin suppressed the expression of several TOR signaling pathway-related genes such as TORC1, ribosomal protein S26A (RPS26A), and ribosomal protein L9A (RPL9A). In addition, parishin significantly diminished the gene expression levels of RPS26A and RPL9A in uth1Δ mutant, as well as in uth1 Δsir2Δ double mutants. Also, parishin remarkably suppressed the TORC1 gene expression in uth1 mutants. These study results indicate that parishin exerts its anti-aging activities by modulating Sir2/Uth1/TOR signaling pathway.¹³¹

Phloridzin

Xiang, L. et al., investigated the anti-aging effects of apple polyphenol, phloridzin, using yeast RLS model. Phloridzin treatment significantly increased the percentage viability of yeast cells exposed to hydrogen peroxide. It was also demonstrated that phloridzin treatment led to increased expression of SOD1/2 and SIRT1 genes as well as the activity of superoxide dismutase enzyme. Altogether, it was suggested that the anti-aging effects of phloridzin are mediated through the regulation of expression of SIRT1 in budding yeast.¹²⁹

Rapamycin

Eukaryotic cells growth and proliferation is regulated by an evolutionarily conserved kinase, the target of rapamycin (TOR). It has been shown that the inactivation of TOR signaling pathway leads to lifespan extension in different eukaryotic model organisms. The eukaryotic nucleolus is rich in ribosomal DNA (rDNA) that is composed of multiple tandem repeats of rRNA genes (100-200 copies rDNA repeats) and the components for ribosome assembly. Inside the nucleolus, the transcription of rDNA produces precursor rRNA that in turn will be processed further and become associated with ribosomal proteins to form preribosomal subunits.

Sir2 is a histone deacetylase enzyme involved in silencing the transcription at the rDNA locus to enhance yeast life span.^145,146 Nearly 50% of the rDNA repeats are maintained in a silent state partly by the Sir2 protein.¹⁴⁷ Using S. cerevisiae yeast as model, it was found that rapamycin treatment cause inhibition of the TORC1 complex, resulting in the increased association of Sir2 with ribosomal DNA (rDNA) in the nucleolus. This association of SIR2 with rDNA reduces homologous recombination between rDNA repeats that causes formation of toxic extrachromosomal rDNA circles. Thus, rapamycin treatment-induced TORC1 inhibition signals the stabilization of rDNA locus by promoting the association of Sir2 with rDNA, thereby extending the RLS in S. cerevisiae.¹⁴⁸

Resveratrol

Mitochondrial dynamics, the balance between mitochondrial fission and fusion, is critical for cell growth and functioning. Replicative senescence in yeast is characterized by the presence of fragmented mitochondria due to mitochondrial fission rather than fusion, indicating altered mitochondrial dynamics. Wang et al. reported that treatment with resveratrol led to a significant reduction in the number of senescent yeast cells with fragmented mitochondria. This indicates that resveratrol’s anti-aging effects possibly occur via modulating the expression of genes associated with mitochondrial dynamics during RLS.¹³⁰

Psoralea corylifolia

P. corylifolia is a medicinal plant widely used in the traditional medicine systems in India and China. Wang et al. showed that the ethanol extract of P. corylifolia significantly extended the RLS of yeast. In particular, the n-hexane fraction of the ethanol extract of P. corylifolia increased the yeast lifespan by 20%. Interestingly, the n-hexane extract of P. corylifolia extended the mean lifespan of the sir2Δfob1Δ double mutant strain, indicating that the n-hexane fraction prolongs the RLS in a Sir2-independent pathway. Treatment with P. corylifolia did not extend the RLS of the tor1Δ mutant, suggesting that Tor1 plays a major role in the extension of RLS by the n-hexane-soluble fraction P. corylifolia.

Furthermore, it was reported that, Corylin and neobavaisoflavone are the active compounds in P. corylifolia that significantly increased the viability and extended the RLS of yeast. In particular, corylin was found to be more effective in enhancing the yeast RLS compared to neobavaisoflavone. It was also shown that corylin treatment promoted RLS of the sir2Δfob1Δ mutant strain but failed to prolong the RLS of the tor1Δ mutant, suggesting that corylin exerts life span-extending effects in a Tor1-dependent pathway. Fascinatingly, under CR conditions, corylin could not extend the RLS. In addition, corylin treatment did not significantly influence the CLS of yeast. Gtr1 in S. cerevisiae encodes a highly conserved GTPase that is necessary for TORC1 activation and amino acid sensing. The G protein complex Gtr1/Gtr2 activates Tor1 in S. cerevisiae in response to signals from amino acids.¹⁴⁹ Furthermore, it was also suggested from docking studies that corylin prolongs the yeast RLS by blocking Gtr1 activation.¹³²

Previous studies by Lee MB et al also demonstrated that green tea extract and berberine could strongly shorten the yeast life span, whereas Pterocarpus marsupium extract and other mixtures containing P. marsupium significantly extended the yeast life span.¹³³ In another study, two sesquiterpene glucosides isolated from the Shenzhou honey peach fruit were also able to extend the RLS of K6001 yeast. Treatment with sesquiterpene glucosides increased the survival rate of yeast under oxidative stress. Besides, treatment with sesquiterpene glucosides could not affect the RLSs of SOD mutant yeast strains with a K6001 background, indicating that the anti-oxidative stress response performs important roles in anti-aging effects of sesquiterpene glucosides.¹²³

Natural products/plant extracts vs. both CLS & RLS

Some natural products have also been demonstrated to extend both RLS and CLS in yeast model. These natural products include curcumin (a diarylheptanoid),¹⁵⁰ Cucurbitacin B (a triterpenoid),¹³⁶ Gentirigeoside B (a dammaren-type triterpenoid glycoside),¹⁵¹ Gentiopicroside (a secoiridoid glycoside),¹⁵² and inkosterone (a phytoecdysteroid).¹⁵³ Table 3 lists the natural compounds, their concentrations, phytochemical class, type of growth medium used for aging experiments, and their anti-aging mechanisms in different S. cerevisiae strain backgrounds.

Curcumin

Curcumin is a biologically active yellow-colored carotenoid compound with potent anti-oxidant and anti-aging properties. Previous studies investigated the replicative and chronological life span-extending effects of curcumin using yeast. It was demonstrated that curcumin significantly increased oxidative stress and enhanced both replicative and chronological life spans of yeast mutants that lacked antioxidant genes (SOD1 and SOD2) and DNA damage repair gene RAD52. Overall, it can be suggested that curcumin exerts anti-aging hormetic effects in yeast.¹⁵⁰

Gentirigeoside B and gentiopicroside

Gentirigeoside B is a triterpenoid glycoside isolated from the Chinese traditional medicinal plant Gentiana rigescens Franch. Xiang, L., et al. reported that gentirigeoside B significantly extended both the replicative and chronological lifespans of yeast. It was also demonstrated that treatment with gentirigeoside B augmented the activity of antioxidant enzymes (including superoxide dismutase, catalase, and glutathione peroxidase) and reduced oxidative stress markers (ROS and MDA), thereby preventing oxidative stress-induced reduction in the viability of yeast cells. Gentirigeoside-B treated cells also showed downregulation of Sch9 and activation of Rim15 and Msn2 proteins. This indicates that the anti-aging potential of gentirigeoside B is due to its ability to inhibit the TORC1/Sch9/Rim15/Msn signaling. However, gentirigeoside B could not enhance the life span of yeast mutant lacking antioxidant genes (sod1∆, sod2∆, cat1∆, and gpx∆) and age-related genes (skn7∆ and uth1∆). Therefore, Xiang, L., et al. suggested that though treatment with gentirigeoside B might have longevity-promoting effects in humans, the effect may not be effective in those with mutations in endogenous antioxidant enzyme genes.¹⁵¹

In another study, gentiopicroside, a secoiridoid glycoside that was also isolated from G. rigescens Franch was reported to extend both the RLS and the CLS of yeast. Gentiopicroside was shown to induce ATG32 gene expression, but could not prolong the RLS and CLS of yeast mutants deficient in ATG32 gene. It was also reported that gentiopicroside enhanced the yeast survival rate under oxidative stress condition by augmenting the activities of enzymatic antioxidants and diminishing the accumulation of ROS and lipid peroxidation. However, gentiopicroside could not affect the RLSs of sod1Δ, sod2Δ, uth1Δ, and skn7Δ. Overall, it can be suggested that gentiopicroside’s anti-aging effects are attributed to its ability to ameliorate autophagy and antioxidative stress response.¹⁵²

Inokosterone

A recent study,⁵⁸ isolated a compound, inokosterone from G. rigescens Franch and demonstrated that inokosterone can extend both the chronological and replicative life spans. The inokosterone was shown to enhance the survival rate of yeast cells by ameliorating the levels of antioxidant enzymes (e.g., SOD) and preventing accumulation of oxidative stress markers (e.g., ROS and MDA levels). Further, inkosterone could alleviate the autophagy (especially mitophagy) in yeast cells. Likewise, the same study also demonstrated that treatment with inkosterone decreased oxidative stress and enhanced autophagy in mammalian cell lines. Therefore, it can be suggested that inkosterone’s anti-aging effects are mediated through the activation of antioxidant stress response and mitophagy.¹⁵³

Emerging trends and future directions

An exponential interest in the pursuit for youthful, vital, and quality of human life have become the key driving forces in the development of novel scientific and technological advancements in the anti-aging field. Recent key developments in anti-aging research include nanocapsulation, nutrigenomics, stem cell therapy, senescent therapy, as well as the application of artificial intelligence (AI). These innovations are useful for forecasting the potential of different anti-aging treatments and to develop personalized anti-aging strategies. Nanocapsulation of anti-aging compounds ensure their sustainable release at target site improving the therapeutic effectiveness. The development of personalized anti-aging nanoformulations might enhance treatment efficacy with minimal side effects.^154,155 Nutrigenomics is an emerging filed that explores how nutrients and dietary factors influence cellular processes. This research focuses on how these factors can modulate age-related gene expression, reduce the risk of age-related diseases, and improve overall quality of life. Understanding gene-diet interactions may allow for the development of precise anti-aging nutritional strategies to promote health and longevity.¹⁵⁶ Stem cell therapy, particularly, the use of mesenchymal stem cells that can differentiate into skin cells, make them potentially restore skin elasticity and combat skin-aging.¹⁵⁷ Senotherapy: There has been a growing interest in the screening, identification, evaluation, and development of potent senotherapeutics including senolytics that selectively kill senescent cells and senostatics/senomorphics that reduce the senescent associated pro-inflammatory phenotype. thereby improving healthspan and potentially reducing the prevalence of chronic conditions.¹⁵⁸ Artificial Intelligence (AI): The revolutionary development of AI algorithms could help in predicting different age-related biomarkers and the progression of age-linked diseases, such as cancer, cardiovascular diseases, neurodegenerative diseases, diabetes, etc.¹⁵⁹ AI's ability to predict diseases related biomarkers offers hope for developing targeted anti-aging therapies and managing the disease risk associated with an aging population.