**Acknowledgements**

*Saccharomyces*

of the RLS assay [6, 43].

properties.

method, as well [15, 45].

popular is spotting test, CLS and RLS assays.

**4. Conclusions**

being smaller than the mother cells of which it is derived. Daughter cells were isolated on a solid growth media and, once they started dividing, all daughter cells were removed. Longo (2012) founded that an individual cells do not divide forever, instead they would stop after a limited number of cells divisions (around of 20–25) and going to a short post-replicative state followed by death cells. The RLS method determines the number of daughters of a single mother cell, which can asexually produce prior to senescence. The mother-daughter cell asymmetry in *S. cerevisiae* cells can be easily observed under the light microscope, allowing the development

As for RLS assay, the *S. cerevisiae* cells inoculated onto 5 mL galactose or glucose liquid medium and further incubated in a shaking incubator for 48 h at the optimal temperature. Subsequently, 1 mL of yeast cells culture was centrifuged and pellet washed with distilled water or phosphate buffer solution (PBS). After counting using a hemocytometer, 4000–5000 cells are plated on agar plates medium containing chemical compounds need to be assay for antiaging activity, and plates were further incubated for 2 days at the optimal temperature. The 40 microcolonies that formed on the agar plates were randomly observed under a microscope, and daughter cells were counted. Recently, *S. cerevisase* yeast cells reported as the most powerfull model organism for RLS analysis [44, 45]. The results of RLS assay usually show in the particular graphic that indicating the viability number from each generations of the yeast cells. If the viability of the yeast cells after compound treatment is higher than control treatment, it is indicating the potential anti-aging

RLS method is reported as having some major weakness which it makes less effective for high-throughput approaches. It is including time-consuming, laborious and relatively intricate in technique due to applying microscopic cells observation prior for plating in the plate medium during assays. Nevertheless, this particular methods will devote precisely quntitatively results and thus could represent the number of yeast cells generation between control and anti-aging compound treatments. To date, some previous studies were reported for using *S. cerevisiae* RLS method to examine anti-aging compound derived from various natural products, including Ganodermasides isolated from mushroom, or Hesperidin from citrus [46, 47]. Current studies were also informed anti-aging assay of some compounds i. e Parishin and Cucurbitacin B using *S. cerevisiae* RLS

Antiaging study in yeast was popular using CR condition which has numerous response to prolong yeast lifespan. Aging pathway in CR belong to pro-and anti-aging pathways. As for pro-aging are including TOR, SCH9, Ras protein, AC, PKA, ethanol accumulation, and apoptotic process. On the other hand, anti-aging pathways are including induction of antioxidative enzymes, sirtuin2, autophagy and adaptive response thorough mitochondrial adaptive ROS signaling. CR condition usually use for positive control, while treatment conducted in non-CR/high 2% glucose medium. There are numerous methods for anti-aging study, which the most

Recently, anti-aging in *S. cerevisiae* research was developed sophisticated, derived from previous sources including natural compound derived from terestrial or aquatic organism. In fact, other sources also potentially developed i.e. semisynthetic or synthetic compound as the preliminary screening for anti-aging

**104**

compounds.

The authors would like to thank Dr. Rika Indri Astuti Department of Biology, IPB University, Indonesia for permitting the pictures.
