**4. Materials and Methods**

#### *4.1. Materials*

The wild-type strain *S. cerevisiae* BY4742 (ATCC®, 201389TM) (*MAT*α *his3*Δ1 *leu2*Δ0 *lys2*Δ0 *ura3*Δ0) was obtained from American Type Culture Collection (Manassas, VA, USA). The culture of yeast reference strain was aliquoted into 10 μL and stored at −80 ◦C. All L-amino acids, yeast nitrogen base w/o amino acids (YNB), ammonium sulfate, peptone, agar and yeast extract, H2DCFDA, 2,4,6-tripyridyl-s-triazine (TPTZ), 2,2- -azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 1,1-diphenyl-2-picrylhydrazyl (DPPH), dimethyl sulfoxide (DMSO), neohesperidin, naringin, hesperidin and hesperetin were bought from Sigma-Aldrich (Shanghai, China). YPD Broth, YPD and other chemicals were from Solebo biotech Co., Ltd. (Beijing, China). The 96-well polystyrene microplates with flat bottom were purchased from Corning incorporated (Kennebunk, ME, USA).

#### *4.2. Lifespan and Yeast Cell Growth Assay*

The determination of chronological lifespan (CLS) of yeast was carried out according to the method of Wu et al. [25] with a moderate modification as follows. In brief, the yeast cells were prepared by transferring a streaked strain from frozen stocks onto YPD agar (0.5% yeast extract/1% peptone/2% dextrose/1.4% agar) plates. After incubating the cells at 30 ◦C for 2 days, a single colony was picked and inoculated into a 1.0-mL YPD liquid medium (1% yeast extract/2% peptone/2% dextrose) in a 10-mL sterilized centrifuge tube (round bottom) and cultured at 30 ◦C for 2 days in a flat incubator at 200 rpm. The 2-day YPD culture was diluted with autoclaved 18 mΩ Milli-Q grade water (1:10) and stored in a refrigerator at 4 ◦C for 2 days. After 2-day incubation at 4 ◦C, 5 <sup>μ</sup>L (<sup>≈</sup> 1 <sup>×</sup> 104 cells) of the diluted culture was transferred to 993 microliter of synthetic-defined (SD) media (Supplementary Table S1, [51]) and maintained at 30 ◦C, 200 rpm for the entire experiment. Compounds in DMSO with several concentrations (2.0 μL) were added at the initial inoculation (0 h). Each experiment was performed at least in triplicate. Cell cultures were incubated at 30 ◦C without replacing the aging medium throughout the experiment. After 2 days of culture in an aging media, the cells reached stationary phase and the first age-point was then taken. Subsequent age-points were taken every 2–4 days. For each age point, 5.0 μL of the mixed culture was pipetted into each well of 96-well flat-bottom microplate. Ninety-five microliter YPD medium was then added to each well. The cell population was monitored with a microplate reader (Varioskan Flash; Thermo Scientific, Waltham, MA, USA) by recording OD660 every 10 min during 24 h.

The survival rate was calculated as follows [25]. Where tOD= 0.3, 2day is the time that OD value of day 2 age-point reaches 0.3 in the outgrowth curves. The initial age-point (day 2) is defined to be 100% viability and the relative survival percent of each successive age-point can be calculated as follows:

$$Vn = \frac{1}{2\frac{\Delta t\_n}{Dt\_n}} \times 100n = days.\ \overline{Dt\_n}\text{ represent the average doubling time.}$$

The survival integral (SI) for each well is defined as the area under the survival curve (AUC) and can be estimated by the formula:

$$\text{SI} = \sum\_{2}^{n} \left( \frac{V\_{n-1} + V\_n}{2} \right) \left( day\_n - day\_{n-1} \right)$$

where *dayn* is the age point, such as days 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20.

#### *4.3. Intracellular ROS Scavenging Ability Assays*

To quantify the intracellular reactive oxygen species (ROS) level of yeast cells grown in standard SD medium with/without the four compounds, the method described in Wu et al. [51] had been referenced. Namely, 2 μL ROS probe H2DCFDA from a fresh 5-mM stock solution in DMSO was added into 1.0 mL yeast aging culture (at day 2) at 30 ◦C for 1 h. The culture was then washed twice in sterile distilled water and suspended in 1.0 mL of 50 mM Tris/Cl buffer (pH 7.5). Twenty microliter of chloroform and 10 μL of 0.1% (*w*/*v*) sodium dodecyl sulfate (SDS) were added, and the cells were incubated at 200 rpm for 30 min to allow the dye to diffuse. The culture was centrifuged at 5000 rpm for 5 min, and the fluorescence of the supernatant was measured using a microplate reader with excitation at 480 nm and emission at 520 nm.

#### *4.4. Antioxidant Activity Assays*

In this study, DPPH, FRAP and ABTS assays were used for in vitro antioxidant capacity evaluation. The DPPH assay was performed according to the method described by Barreca et al. [53]. An aliquot of each sample (0.5 mL) was mixed with 75 μM (3.5 mL) of DPPH in methanol to a final volume of 4.0 mL. After reacting for 30 min without light, the absorption of the mixture was detected at a wavelength of 517 nm. The inhibition percentage of radical scavenging activity was the DPPH value. The FRAP assay was carried out according to Hungder et al. [54] with some modifications. A 0.2 mL aliquot of the sample was mixed with 3.8 mL of FRAP reagent (0.3 mol/L acetate buffer (pH 3.6), 10 mmol/L TPTZ solution, and 20 mmol/L ferric chloride (FeCl3) were mixed (10:1:1, volume ratio)). After 30 min, the absorbance was detected at wavelength of 593 nm. And the ABTS assay was followed the method of Mnb et al. [55] with few modifications. The ABTS radical cation (ABTS •+) was generated by reaction of 176 μL of potassium persulfate solution (140 mM) and 10 mL aqueous ABTS solution (7 mM) under the condition of no light for 12–16 h. Then it was diluted with methanol to an absorption value of 0.7 ± 0.02 units at 734 nm. 0.1 mL sample was added to 4.9 mL ABTS reagent. The absorbance was measured at a wavelength of 734 nm after 10 min reaction. All absorbance values were determined by using the UV–VIS spectrophotometer (PerkinElmer Lambda 25 UV/VIS, Waltham, MA, USA). Antioxidant values were calculated by standard curve method and expressed as trolox equivalents (TE mg/g DW).

### *4.5. Extracellular pH Detection*

The culture process of yeast in the early stage was almost the same as the method described in the part of "Lifespan and yeast cell growth assay". The specific operation was as follows. The yeast cells were prepared by transferring the yeast strain from frozen stocks onto YPD agar plates. After incubating the cells at 30 ◦C for 2 days, a single colony was picked and inoculated into a 1.0-mL YPD liquid medium in a 10-mL sterilized centrifuge tube and cultured at 30 ◦C for 2 days in a flat incubator at 200 rpm. The 2-day YPD culture was diluted with autoclaved 18 mΩ Milli-Q grade water (1:10) and stored in a refrigerator at 4 ◦C for 2 days. After 2-day incubation at 4 ◦C, 10 <sup>μ</sup>L (<sup>≈</sup> 2 <sup>×</sup> 104 cells) of the diluted culture was transferred to 1986 μL of SD media and maintained at 30 ◦C, 200 rpm for 2/10/20 days. Various compounds in DMSO (4.0 μL) were add to the medium to a final concentration of 10 μM at initial inoculation (0 h). Then 1mL of the 2/10/20-day SD culture was added to 19 mL fresh YPD liquid medium. The pH was tested every five minutes using a pH meter while the yeast was cultured at 30 ◦C in a shaker at 200 rpm for the whole detection. Each experiment was performed at least in triplicate.
