Discovery of plant extracts that greatly delay yeast chronological aging and have different effects on longevity-defining cellular processes

We discovered six plant extracts that increase yeast chronological lifespan to a significantly greater extent than any of the presently known longevity-extending chemical compounds. One of these extracts is the most potent longevity-extending pharmacological intervention yet described. We show that each of the six plant extracts is a geroprotector which delays the onset and decreases the rate of yeast chronological aging by eliciting a hormetic stress response. We also show that each of these extracts has different effects on cellular processes that define longevity in organisms across phyla. These effects include the following: 1) increased mitochondrial respiration and membrane potential; 2) augmented or reduced concentrations of reactive oxygen species; 3) decreased oxidative damage to cellular proteins, membrane lipids, and mitochondrial and nuclear genomes; 4) enhanced cell resistance to oxidative and thermal stresses; and 5) accelerated degradation of neutral lipids deposited in lipid droplets. Our findings provide new insights into mechanisms through which chemicals extracted from certain plants can slow biological aging.


INTRODUCTION
The budding yeast Saccharomyces cerevisiae is a unicellular eukaryote amenable to comprehensive molecular analyses [1][2][3]. The development of various methods of such analyses for S. cerevisiae has enabled to uncover mechanisms underlying complex biological processes within individual yeast cells and their populations [1,4,5]. In addition, S. cerevisiae has relatively short and easy measurable replicative and chronological lifespans [6][7][8][9][10][11][12][13]. Due to these beneficial properties as a model organism for studying mechanisms of aging and longevity, S. cerevisiae has been used for the discovery of genes that slow cellular aging and increase healthy lifespan not only in S. cerevisiae and other yeasts but also in multicellular eukaryotes [6,7,9,11,[14][15][16]. Furthermore, using S. cerevisiae as a model organism for elucidating mechanisms of cellular aging, several nutrient-and energy-sensing signaling pathways have been revealed; these pathways coordinate an evolutionarily conserved array of longevitydefining cellular processes not only in S. cerevisiae and other yeasts but also in eukaryotes across phyla [9,11,[17][18][19][20]. Moreover, S. cerevisiae has been a model organism employed for uncovering several low molecular weight molecules that slow aging and extend healthy lifespan in various multicellular eukaryotes [10,[21][22][23][24][25][26][27]. All these studies employing S. cerevisiae as a model organism have provided evidence that the main features of biological aging have been conserved in the course of evolution [6,9,11,18,21,[28][29][30][31].
Our research is aimed at using S. cerevisiae as a model organism to discover chemical compounds that can slow aging and delay the onset of age-related diseases in evolutionarily distant eukaryotic organisms. Some of such geroprotective compounds have been previously revealed in natural products extracted from certain plants [25,32,33]. As a first step towards uncovering novel aging-delaying chemical compounds of plant origin, we conducted a screen Oncotarget 16543 www.impactjournals.com/oncotarget for plant extracts (PEs) that can extend yeast chronological lifespan (CLS). Our screen revealed six PEs that increase yeast CLS considerably more efficiently than any of the longevity-extending chemical compounds yet described. We show that each of these PEs decelerates yeast chronological aging and has different effects on several longevity-defining cellular processes.

A screen for PEs that can extend longevity of chronologically aging yeast
We screened a library of PEs for extracts that can increase yeast CLS. This library includes 35 different PEs of known origin and properties (Tables 1 and 2, respectively). To perform the screen for lifespan-extending PEs, we used a robust assay for measuring yeast CLS. This assay was similar to the one described previously [34], but the wildtype strain BY4742 was cultured in the synthetic minimal YNB medium initially containing 2% glucose (instead of the nutrient-rich YEP medium supplemented with 0.5% glucose). Yeast cells cultured on 2% glucose are not limited in calorie supply; these cells age chronologically under so-called non-caloric restriction (non-CR) conditions that accelerate aging in different yeast genetic backgrounds, including BY4742 [6,10,11].
In our screen for longevity-extending PEs, each PE from the library was added to growth medium at the time of cell inoculation at a final concentration in the 0.02% to 1.0% range. Some PEs from the library did not alter the mean and maximum CLS of yeast under non-CR conditions at any concentration examined; among these PEs were PE9, PE13, PE16, PE22, PE28 and PE36 ( Figure S2- Figure S5). Many PEs from the library shortened the mean and/ or maximum CLS of yeast under non-CR conditions at final concentrations ranging from 0.08% to 1.0%; among these PEs were PE1-PE3, PE7, PE10, PE11, PE14, PE15, PE17-PE20, PE24, PE25, PE27, PE29-PE35 and PE37 (Figure S1- Figure S5). 6 of the 35 PEs from the library significantly increased both the mean and maximum CLS of yeast under non-CR conditions if added at final concentrations ranging from 0.04% to 1.0% (Figure S1- Figure S3). A group of these longevityextending PEs included the following extracts: 1) 0.5% PE4 from Cimicifuga racemosa ( Figure 1A, Figure 3A and 3B, Figure S1); 2) 0.5% PE5 from Valeriana officinalis L. (Figure 1B, Figure 3A and 3B, Figure S1); 3) 1.0% PE6 from Passiflora incarnata L. (Figure 1C, Figure 3A and 3B, Figure S1); 4) 0.3% PE8 from Ginkgo biloba ( Figure 1D, Figure 3A and 3B, Figure S1); 5) 0.1% PE12 from Apium graveolens L. (Figure 1E, Figure 3A and 3B, Figure S2); and 6) 0.1% PE21 from Salix alba ( Figure 1F, Figure 3A and 3B, Figure S3). None of the six lifespan-prolonging PEs affected growth rates in logarithmic (L) and postdiauxic (PD) phases or impacted the maximum cell density in stationary (ST) phase of yeast cultures under non-CR conditions on 2% glucose ( Figure S6). Thus, the observed lifespan extension by each of these PEs is unlikely to be caused by its ability to decrease growth rate or to make yeast more resistant to toxic substances accumulated during culturing in the synthetic minimal YNB medium.
For each of the six lifespan-prolonging PEs, the longevity-extending efficacy under CR conditions is significantly lower than that under non-CR conditions Chronologically aging yeast grown under CR conditions on 0.5% glucose are known to live longer than yeast cultured under non-CR conditions on 2% glucose. Such ability of the CR diet to extend CLS has been reported for yeast cultured in media of various nutrient compositions [6,10,11]. We found that, if the CR diet is administered by culturing yeast in the YNB medium initially containing 0.5% glucose, it significantly increases both the mean and maximum CLS of S. cerevisiae (Figure 2A-2C). We discovered that 0.5% PE5 and 0.1% PE21 (but not 0.5% PE4, 1.0% PE6, 0.3% PE8 or 0.1% PE12) extend the mean CLS of yeast grown under CR conditions ( Figure 2D-2I, Figure 3C). We also revealed that 0.5% PE5, 1.0% PE6 and 0.1% PE21 (but not 0.5% PE4, 0.3% PE8 or 0.1% PE12) extend the maximum CLS of yeast grown under CR conditions ( Figure 2D-2I, Figure 3D). Akin to their effects under non-CR conditions, none of the six lifespan-prolonging PEs influenced growth rates in L and PD phases or altered the maximum cell density in ST phase of yeast cultures under CR conditions on 0.5% glucose ( Figure S7). Importantly, each of the six lifespan-prolonging PEs extended both the mean and maximum CLS of yeast cultures under non-CR conditions on 2% glucose to a significantly higher extent than those of yeast under CR at 0.5% glucose ( Figure 3A-3F). This observation indicates that each of these PEs could mimic the longevityextending effect of CR.
Each of the six longevity-extending PEs is a geroprotector which delays the onset and slows the progression of yeast chronological aging by eliciting a hormetic stress response As we found, PE4, PE5, PE6, PE8, PE12 and PE21 greatly extend the mean CLS of yeast cultured under non-CR conditions ( Figure 1A, Figure 3A and 3B, Figure S1-S3). Mean lifespan is believed to be directly proportional to the survival rates of organisms in the population during development and maturity stages of organismal aging; mean lifespan is likely to be under control of certain extrinsic (environmental) factors [35][36][37][38]. Thus, it is conceivable that PE4, PE5, PE6, PE8, PE12 and PE21 decrease the extrinsic rate of yeast chronological aging prior to cell entry into quiescence or senescence. www.impactjournals.com/oncotarget
Our analysis of the Gompertz mortality function further validated the above conclusion that PE4, PE5, PE6, PE8, PE12 and PE21 significantly reduce the rate of yeast chronological aging. Indeed, we found that each of these longevity-extending PEs causes a substantial decrease in slope of the Gompertz mortality rate (also known as mortality rate coefficient α) and a considerable increase in the mortality rate doubling time (MRDT) ( Figure 4A-4G). Such changes in the values of α and MRDT are characteristic of interventions that decrease the rate of progression through the process of biological aging [37,[41][42][43][44].
Noteworthy, our analyses of how different concentrations of PE4, PE5, PE6, PE8, PE12 and PE21 impact yeast longevity under non-CR conditions revealed that each of them causes a so-called "hormetic" stress response in chronologically aging yeast with respect to longevity. Indeed, the dose-response curves (i.e. the curves that reflect relationships between PE concentrations and mean or maximum CLS) for PE4, PE5, PE8, PE12 and PE21 were inverted U-shaped, whereas the dose-response curve for PE6 was J-shaped ( Figure S1-S3). Such nonlinear and biphasic dose-response curves denote a hormetic kind of stress response, in which 1) lower (hormetic) concentrations of a chemical compound increase the survival of a cell or an organism by stimulating biological processes that allow to maintain cellular or organismal stress at a level which is below a threshold of toxicity; while 2) higher concentrations of this chemical compound decrease the survival of a cell or an organism by creating stress which exceeds such threshold [25,[45][46][47][48].
We found that each of the six lifespan-extending PEs stimulates coupled mitochondrial respiration, which was monitored by measuring the rate of oxygen consumption by yeast cells. PE4, PE8 and PE12 decreased the extent to which such respiration declined in ST-phase cultures ( Figure 5A, 5D and 5E), whereas PE5, PE6 and PE21 considerably increased the rate of mitochondrial respiration in yeast during PD and ST growth phases ( Figure 5B, 5C and 5F). We also found that each of the six lifespan-extending PEs sustains healthy populations of functional mitochondria that exhibit high mitochondrial membrane potential (ΔΨ m ). PE4, PE8 and PE12 substantially reduced the extent to which ΔΨ m declined during PD and ST growth phases ( Figure 6A, 6D and 6E; Figure S8 and S9), whereas PE5, PE6 and PE21 completely prevented such decline ( Figure  6B, 6C and 6F; Figure S8 and S9).
PE4, PE5, PE6, PE8, PE12 and PE21 also caused significant changes in the age-related chronology of intracellular ROS, which in yeast and other organisms are known to be formed mainly as by-products of mitochondrial respiration [68,69]. Each of these PEs decreased the extent to which the intracellular concentration of mitochondrially The six lifespan-extending PEs differently influence the extent of an age-related oxidative damage to cellular proteins, membrane lipids, mitochondrial and nuclear genomes A body of evidence supports the following view on the relationships between cellular ROS, oxidative molecular damage and aging in organisms across phyla: 1) if cellular concentrations of ROS exceed a threshold of toxicity, ROS cause oxidative damage to proteins, lipids and DNA; 2) oxidative damage to each kind of these macromolecules accumulates with age; and 3) cumulative oxidative damage to the different kinds of macromolecules is one of the major causes of aging [53,54,[69][70][71][72][73][74][75]. We therefore examined how PE4, PE5, PE6, PE8, PE12 and PE21 impact the extent of oxidative damage to proteins, lipids and DNA in chronologically aging yeast cultured under non-CR conditions on 2% glucose.
We found that each of the six lifespan-extending PEs delays an age-dependent rise in the extent of oxidative Furthermore, PE5, PE6, PE8, PE12 and PE21 (but not PE4) caused a significant reduction in the levels of oxidatively damaged membrane lipids; such reduction was observed late in ST phase, on days 3 and/or 4 of culturing ( Figure 8H-8L).
Moreover, PE4, PE5, PE6, PE8, PE12 and PE21 decreased the frequencies of spontaneous point mutations in the RIB2 and RIB3 genes of mitochondrial DNA (mtDNA) ( Figure 9A-9F) -likely due to a reduced extent of oxidative damage to mtDNA in yeast cells exposed to any of these PEs. Such inhibitory effects of the six lifespan-extending PEs on oxidative damage to mtDNA was observed late in ST phase, on day 4 of culturing.
We also revealed that PE12 and PE21 (but not PE4, PE5, PE6 or PE8) caused a significant reduction in the frequencies of spontaneous point mutations in the      (Figure 9K and 9L) -possibly due to a decreased degree of oxidative damage to nDNA in yeast cells grown in the presence of PE12 or PE21. Such inhibitory effects of PE12 or PE21 on oxidative damage to nDNA was also seen late in ST phase, on day 4 of culturing.
The six lifespan-extending PEs differently influence the resistance of chronologically aging yeast to chronic oxidative and thermal stresses A body of evidence implies that the development of resistance to chronic (long-term) oxidative and/or thermal stresses can extend longevity in organisms across phyla, including yeast [6, 9, 10, 11, 34, 46-48, 68, 76-80]. We therefore assessed how PE4, PE5, PE6, PE8, PE12 and PE21 influence the abilities of chronologically aging yeast cultured under non-CR conditions to resist chronic oxidative and thermal stresses.
Chronic oxidative stress was administered by recovering yeast cells progressing through L, PD or ST phases of growth/culturing in liquid YNB medium initially containing 2% glucose, spotting these cells on solid YEP medium with 2% glucose and 5 mM hydrogen peroxide, and incubating them for 3 days. We found that PE6, PE12 and PE21 significantly increase cell resistance to chronic oxidative stress in yeast cultures progressing through L, PD and ST phases ( Figure 10A and 10B). PE4, PE5 and PE8 enhanced the ability of cells to resist chronic oxidative stress only in yeast cultures progressing through ST phase, but did not alter such ability during L and PD phases ( Figure 10A and 10B).
Chronic thermal stress was administered by recovering yeast cells progressing through L, PD or ST phases of growth/culturing in liquid YNB medium initially containing 2% glucose, spotting these cells on solid YEP medium with 2% glucose and incubating at 60 o C for 60 min, and then transferring plates with these cells to 30 o C and incubating at this temperature for 3 days. We found that PE6, PE8, PE12 and PE21 increase cell resistance to chronic thermal stress only in yeast cultures progressing through ST phase ( Figure 11A and 11B). In contrast, each of these four lifespan-extending PEs weakened the ability of cells to resist chronic thermal stress during L and PD phases ( Figure 11A and 11B). Furthermore, neither PE4 nor PE5 altered cell resistance to chronic thermal stress in yeast cultures progressing through ST phase ( Figure 11A and 11B). PE4 did not affect the ability of cells to resist chronic thermal stress also during L and PD phases, whereas PE5 reduced such ability in yeast cultures progressing through these two phases ( Figure 11A and 11B).

Each of the six lifespan-extending PEs causes rapid degradation of neutral lipids deposited in lipid droplets
Triacylglycerols and steryl esters are uncharged (and therefore are called "neutral" or "nonpolar") classes of lipids that can be found in cells of all eukaryotic organisms [81][82][83]. After being initially synthesized in the endoplasmic reticulum and then deposited in lipid droplets (LDs), these two highly hydrophobic lipids can undergo lipolytic degradation to provide substrates for the synthesis of phospholipids and sphingolipids [82,[84][85][86][87]. Emergent evidence supports the view that the biosynthesis, storage and lipolysis of neutral lipids are longevity assurance processes; importantly, it has been shown that these processes can be controlled by certain dietary and pharmacological interventions known to delay aging in various eukaryotes, including yeast [6,23,34,. We therefore used live-cell fluorescence microscopy to examine how PE4, PE5, PE6, PE8, PE12 and PE21 influence the age-related dynamics of changes in the intracellular concentration of neutral lipids confined to LDs in chronologically aging yeast grown under non-CR conditions.
We found that each of the six lifespan-extending PEs elicits rapid age-related decline in the number of yeast cells exhibiting LDs (Figures 12A-12F; Figure S10 and S11). In contrast, no significant changes in the number of cells with LDs were seen in yeast progressing through L, PD and ST phases of culturing in medium without a PE (Figure 12A-12F; Figures S10 and S11). These findings demonstrate that in chronologically aging yeast grown under non-CR conditions, each of the six lifespanextending PEs causes rapid lipolytic degradation of neutral lipids stored in LDs.

DISCUSSION
In this study, we performed a screen for PEs capable of extending longevity of the chronologically aging yeast S. cerevisiae. Our screen revealed six PEs (which we call PE4, PE5, PE6, PE8, PE12 and PE21) that can significantly increase yeast CLS. We demonstrated that each of these PEs is a geroprotector which delays the onset and slows the progression of yeast chronological aging by eliciting a hormetic stress response. We provided evidence that each of these geroprotective PEs has different effects on cellular processes known to define longevity in organisms across phyla. Such effects include the following: 1) amplified mitochondrial respiration and membrane potential; 2) increased or decreased concentrations of ROS; 3) reduced oxidative damage to cellular proteins, membrane lipids, and mitochondrial and nuclear genomes; 4) enhanced cell resistance to oxidative and thermal stresses; and 5) accelerated degradation of neutral lipids deposited in LDs ( Figure 13). These findings provide important new insights into mechanisms through which some chemical compounds of plant origin can slow biological aging.

Each of the six longevity-extending PEs increases lifespan more efficiently than any lifespanprolonging chemical compound currently known
Our findings imply that the efficiency of longevity extension by PE4, PE5, PE6, PE8, PE12 or PE21 greatly exceeds that for any of the 42 chemical compounds known to increase lifespan in yeasts, filamentous fungi, nematodes, fruit flies, daphnias, mosquitoes, honey bees, fishes, mammals and cultured human cells (Table S1). Indeed, under non-CR conditions these longevity-extending PEs increase the mean and maximum CLS of yeast by 145%-475% and 80%-369%, respectively (Figure 3A and 3B; Table S1); the corresponding rows in Table S1 are highlighted in yellow. In contrast, any of the 42 currently known lifespan-extending chemical compounds has been shown to extend cellular and/or organismal lifespan in evolutionarily distant eukaryotes much less efficiently, within the 5% to 75% range (Table S1) [references 1-27, 30-57, 59-61 for Table S1]. Only two chemical compounds, spermidine under non-CR conditions and lithocholic acid under CR-conditions, have been reported to exhibit the lifespan-extending efficiencies that are comparable to those for PE4, PE5, PE6, PE8 and PE12 (Table S1) [references 28, 29, 58 for Table S1]. Specifically, both these pharmacological interventions were demonstrated to www.impactjournals.com/oncotarget   Arrows pointing at boxes with the terms of longevity-defining cellular processes denote activation of these processes, T bars denote inhibition of these processes, whereas lines with filled circles denote change in the age-related chronology of intracellular ROS. The thickness of such arrows, T bars and lines with filled circles correlates with the extent to which a PE activates, inhibits or alters the age-related chronology (respectively) of a particular longevity-defining cellular process. Arrows and T bars pointing at boxes with the term "AGING" denote acceleration or deceleration (respectively) of yeast chronological aging. www.impactjournals.com/oncotarget increase the RLS and/or CLS of yeast and human peripheral blood mononuclear cells by 83%-200%; the corresponding rows in Table S1 are highlighted in green. Of note, PE21 appears to be the most potent longevity-extending pharmacological intervention presently known. It increases the mean and maximum CLS of yeast by 475% and 369%, respectively (Figure 3A and 3B; Table S1).

Future perspectives
In the future, it would be important to further explore the following key aspects of the mechanisms through which each of the six longevity-extending PEs slows biological aging.
First, it is intriguing to identify the individual chemical compounds responsible for the ability of each of these PEs to delay the onset and decrease the rate of yeast chronological aging. Such identification is already underway in our laboratory; of note, it is conceivable that only some combinations of certain chemicals composing these PEs (but not individual chemical compounds per se) can be responsible for their extremely high efficiencies as aging-delaying interventions.
Second, it is interesting to elucidate how genetic interventions that impair any of the few nutrient-and energysensing signaling pathways known to define longevity of chronologically aging yeast [6,10,11,23] influence the extent to which each of the six longevity-extending PEs can slow aging. These studies may allow to identify protein components of the longevity-defining signaling pathways that are targeted by each of the PEs. These studies may also reveal that certain combinations of these PEs and genetically impaired components of pro-aging signaling pathways exhibit additive or synergistic effects on the efficiencies of lifespan and healthspan extensions.
Third, it is important to investigate how various combinations of the six longevity-extending PEs with each other and with presently known aging-delaying chemical compounds alter the extent of CLS extension in yeast. These studies may identify such combinations of various pharmacological interventions that impose substantial additive or synergistic effects on the efficiencies with which organismal lifespan and healthspan can be prolonged.
Fourth, our ongoing studies indicate that the six longevity-extending PEs also extend longevities of other eukaryotic model organisms, delay the onset of agerelated diseases and/or exhibit anti-tumor effects. In this regard, it needs to be mentioned that genetic, dietary and pharmacological interventions known to delay aging in yeast and other eukaryotes have been shown to selectively kill cultured human cancer cells and/or decrease the incidence of cancer [29,88,[109][110][111][112][113][114][115][116][117][118][119][120]. The challenge for the future is to define mechanisms through which the six geroprotective PEs prolong healthy lifespan and decelerate tumorigenesis.

Yeast strains, media and growth conditions
The wild-type strain Saccharomyces cerevisiae BY4742 (MATa his3D1 leu2D0 lys2D0 ura3D0) from Thermo Scientific/Open Biosystems was grown in a synthetic minimal YNB medium (0.67% Yeast Nitrogen Base without amino acids) initially containing 2% or 0.5% glucose and supplemented with 20 mg/l histidine, 30 mg/l leucine, 30 mg/l lysine and 20 mg/l uracil. Cells were cultured at 30°C with rotational shaking at 200 rpm in Erlenmeyer flasks at a "flask volume/medium volume" ratio of 5:1.

CLS assay
A sample of cells was taken from a culture at a certain day following cell inoculation and PE addition into the medium. A fraction of the sample was diluted in order to determine the total number of cells using a hemacytometer. Another fraction of the cell sample was diluted and serial dilutions of cells were plated in duplicate onto YEP (1% yeast extract, 2% peptone) plates containing 2% glucose as carbon source. After 2 d of incubation at 30°C, the number of colony forming units (CFU) per plate was counted. The number of CFU was defined as the number of viable cells in a sample. For each culture, the percentage of viable cells was calculated as follows: (number of viable cells per ml/total number of cells per ml) × 100. The percentage of viable cells in mid-logarithmic growth phase was set at 100%.

A screen for PEs that can extend yeast CLS
CLS analysis in the presence of various PEs was performed as described above. A 20% stock solution of each PE in ethanol was made on the day of adding this PE to cell cultures. For each PE, the stock solution was added to growth medium with 2% glucose immediately following cell inoculation into the medium. The final concentration of each PE in the medium was 0.02%, 0.04%, 0.06%, 0.08%, 0.1%, 0.3%, 0.5% or 1.0%.

Statistical analysis
Statistical analysis was performed using Microsoft Excel's (2010) Analysis ToolPack-VBA. All data are presented as mean ± SEM. The p values for comparing the means of two groups (using an unpaired two-tailed t test) and survival curves (using a two-tailed t test) were calculated with the help of the GraphPad Prism statistics software.