Antiaging of Cucurbitane Glycosides from Fruits of Momordica charantia L.

Methanol extracts of Momordica charantia L. fruits are extensively studied for their antiaging activities. A new cucurbitane-type triterpenoid (1) and nine other known compounds (2–10) were isolated, and their structures were determined according to their spectroscopic characteristics and chemical derivatization. Biological evaluation was performed on a K6001 yeast bioassay system. The results indicated that all the compounds extended the replicative lifespan of K6001 yeast significantly. Compound 9 was used to investigate the mechanism involved in the increasing of the lifespan. The results indicated that this compound significantly increases the survival rate of yeast under oxidative stress and decreases ROS level. Further study on gene expression analysis showed that compound 9 could reduce the levels of UTH1 and SKN7 and increase SOD1 and SOD2 gene expression. In addition, it could not extend the lifespan of the yeast mutants of Uth1, Skn7, Sod1, and Sod2. These results demonstrate that compound 9 exerts antiaging effects via antioxidative stress and regulation of UTH1, SKN7, SOD1, and SOD2 yeast gene expression.

Aging is a dominating risk factor for age-related diseases, including cancer, metabolic disease, cardiovascular disease, and neurodegenerative illnesses [11]. As the aging population is increasing dramatically throughout the world, aging has drawn great attention because of huge expenses for medical care and serious consequences of the related diseases. Interventions that delay aging were found to have a greater effect on the quality of life compared with disease-specific approaches [12]. In our previous studies [13][14][15][16][17], a yeast mutant K6001 was employed in the bioassay system, and ganodermasides A-D, phloridzin, nolinospiroside F, and parishin with significant antiaging potential from natural sources were obtained.

Material and Methods
2.1. General. The chemical reagents used were of HPLC grade and purchased from TEDIA (Rhode Island, USA). The others were of analytical grade and obtained from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). The preparative HPLC system was equipped with two ELITE P-230 pumps and an UV detector. Optical rotations were determined on a JASCO P-1030 digital polarimeter. High-resolution ESI-TOF-MS analyses were performed on an Agilent Technologies 6224A Accurate-Mass TOF LC/ MS system (Santa Clara, CA, USA). Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker AV III-500 spectrometer (Bruker, Billerica, MA, USA). Column chromatography was performed over the silica gel (200-300 mesh, Yantai Chemical Industry Research Institute, Yantai, China) or reversed phase C18 (Octadecylsilyl, ODS) silica gel (Cosmosil 75C 18 -OPN, Nacalai Tesque, Japan).

Acid Hydrolysis and Sugar
Analysis of Compound 1. The absolute configuration of sugar moiety of compound 1 was determined according to a previously reported method [22,23]. Briefly, compound 1 (0.5 mg) in anhydrous 2.0 M HCl in MeOH (1 mL) was heated at 80°C with reflux for 4 h. The reaction solution was evaporated and partitioned between chloroform and water. The residue of aqueous layer was heated with 0.5 mg L-cysteine methyl ester in pyridine (200 μL) at 60°C for 1 h; then, o-tolyl isothiocyanate dissolved in 100 μL pyridine (7 mg/mL) was added to the reaction mixture and further reacted at 60°C for 1 h. After that, the reaction mixture was dried and analyzed by LC/HRESIMS with the following conditions: Agilent Extend C18 column (3.5 μm, 3.0 × 100 mm); DAD detection, 210 nm; t = 0 min The allose thiocarbamate standards were prepared in the same procedure. Given that L-allose is limitedly available, the retention time of L-allose thiocarbamate derivative was obtained by reacting D-allose with D-cysteine methyl ester. The basis of this approach is the fact that the t R values of D-and L-enantiomers are reversed when D-cysteine methyl ester is used [22].
2.5. Lifespan Assay. The bioassay method was performed as described in a previous study [13]. Briefly, K6001 or mutants with K6001 background were grown on a YPGalactose medium consisting of 3% galactose, 2% hipolypeptone, and 1% yeast extract or on a YPGlucose medium containing 2% glucose instead of galactose. Agar plates were prepared by adding 2% agar to the medium. For screening, the K6001 yeast strain was first incubated in the galactose medium for 24 h with shaking and then centrifuged. The yeast pellet was washed with PBS three times. The cells were then diluted and counted using a hemocytometer, and approximately 4000 cells were plated on glucose agar plates containing different concentrations of samples. The plates were stored in an incubator at 28°C. After 48 h, the yeast cells in the plates were observed with a microscope. For each plate, 40 colonies were selected randomly, and the number of their daughter cells was counted and analyzed.
2.6. Antioxidative Stress Method. Antioxidative stress assay was performed as previously described with minor modification [16]. BY4741 yeast was inoculated in 5 mL of YPGlucose medium and cultured at 28°C with shaking for 24 h. The yeast cells at 0.1 OD 600 were transferred in 20 mL of new YPGlucose medium and incubated with compound 9 at 1 and 3 μM or resveratrol (Res, positive control) at 10 μM for 12 h.
For the first method, 5 μL aliquot after double dilution from each group was dropped in the same YPGlucose agar plate mixed with 9 mM H 2 O 2 , and the plate was incubated at 28°C for 4 days. The growth rates of the yeast cells in different groups were compared and photographed.
Another antioxidative stress assay was used to validate the accuracy of the experiment. Approximately 200 cells mixed with the test samples were spreaded on YPGlucose agar plates with or without 5 mM H 2 O 2 and cultured at 28°C for 48 h. The survival rates of the sample groups were counted and compared with those of the control group.

Determination of ROS Level in Yeast.
The ROS assay procedure was the same with a previous study [17]. BY4741 yeast cells were cultured as described in the experiment above and incubated with compound 9 at 1 or 3 μM for 23 h. Changes in intracellular ROS levels of the yeast were determined using an ROS assay kit (Beyotime, Jiangsu, China) and a fluorescent plate reader (Spectra Max M2, Molecular Devices, San Francisco, CA, USA). A total of 1 mL of cultured broth was obtained, treated with 10 μM DCFH-DA at 28°C in dark, and then shaken by vortexing at 160 rpm at 15 min intervals for 1 h. The yeast cells were subsequently washed with PBS, and their DCF fluorescence was measured by a fluorescent plate reader at excitation and emission wavelengths of 488 and 525 nm, respectively.
2.9. Statistical Analysis. One-way analysis of variance was performed using GraphPad Prism biostatistics software (San Diego, CA, USA) to analyze the data. Significant differences were compared by two-tailed multiple t-tests with Student-Newman-Keuls test. Data were expressed as means ± SEM of triplicate experiments. A P < 0 05 was considered statistically significant.  (Figure 2, in bold bonds). In the HMBC spectrum, these partial structures were connected to yield the following gross structures: H-3 to C-5; H-6 to C-5; H-8 to C-9; H-10 to C-9; CH 3 -18 to C-12, C-13, C-14, and C-17; H-19 to C-9, C-10, C-11, and −OCH 3 ; CH 3 -28 to C-3, C-4, and C-29; CH 3 -29 to C-4, C-5, and C-28; CH 3 -30 to C-8, C-13, C-14, and C-15; H-23 to C-25; H-24 to C-25, C-26, and C-27; H-26 to C-24; CH 3 -27 to C-24; and −OCH 3 to C-19. The signals of H-3 to C-1 ′ of allose and anomeric proton H-1 ′ of allose to C-3 in the HMBC indicated the location of the sugar moiety ( Figure 2). The β anomeric configuration of allose was determined from its coupling constant J (7.8 Hz) of anomeric protons (δ H 5.52). The absolute configuration of the sugar moiety was further confirmed by the degradation of compound 1 and through the comparison of the retention time of its aldose thiocarbamate derivative (t R = 9.287 min) with those of the following aldose thiocarbamate standards: L-cysteine-D-allose (t R = 9.127 min) and D-cysteine-D-allose (t R = 7.460 min).

Compound 9 Improves the Oxidative Resistance and
Decreases ROS Production of Yeast. Studies on mechanism of action were conducted with compound 9 because of its abundance and good activity. Oxidative stress is one of the primary causes of aging, as indicated in various model organisms [24]. Therefore, the effect of compound 9 on the oxidative resistance of yeast was first tested. The growth of yeast cells was inhibited at 9 mM H 2 O 2 , whereas incubation with compound 9 at 1 or 3 μM remitted the inhibition (Figure 4(a)). The effect was further confirmed in another assay. As shown in Figure 4(b), the survival rate of the control group was 48.31% ± 4.94%, whereas that in the experimental groups increased to 71.21% ± 2.75% (compound 9 at 1 μM, P < 0 5) and 68.73% ± 5.89% (compound 9 at 3 μM, P < 0 5). The experiments indicated that compound 9 enhances the oxidative resistance of yeast cells. Furthermore, we detected the ROS level of yeast after administration compound 9 at 1 and 3 μM. As we expected, the ROS level of yeast in the resveratrol and compound 9 groups were significantly decreased compared with the control  Figure 5: Effects of compound 9 on SOD1 (a), SOD2 (b), UTH1 (c), and SKN7 (d) yeast gene expression. The gene levels of BY4741 yeast cells were tested after treated with compound 9 at 1 and 3 μM. Compound 9 significantly increased SOD1 and SOD2 yeast gene level at 12 h and inhibited UTH1 and SKN7 yeast gene expression at 24 h. Amounts of the mRNA above were normalized to that of TUB1. The results were displayed as mean ± SEM for three independent experiments ( * P < 0 05, * * P < 0 01, and * * * P < 0 001, compared with the control group). group (Figure 4(c), P < 0 01, P < 0 01, and P < 0 05), respectively. These results suggested that compound 9 extended the replicative lifespan via inhibition of oxidative stress.
3.5. Compound 9 Extends Yeast Lifespan via Modification of UTH1, SKN7, SOD1, and SOD2 Gene Expression. It is well known that antioxidative stress is one of mechanisms of action for antiaging. UTH1 gene essentially takes part in oxidative stress regulation, and deletion of UTH1 gene will lead to extend the replicative lifespan of yeast [25]. SKN7 is upstream gene and is a stress response transcription factor in Saccharomyces cerevisiae [26]. Superoxide dismutases (SOD) are major ROS scavenging enzymes and can convert superoxide anion to hydrogen peroxide [24]. Real-time PCR analysis was performed to examine the molecular mechanism of compound 9-mediated lifespan extension. The significant gene expression reduction or increase of UTH1, SKN7, SOD1, and SOD2 was observed in the compound 9 treatment groups ( Figure 5). These results suggested that compound 9 produced antiaging effect via regulation UTH1, SKN7, SOD1, and SOD2 yeast gene expression.
3.6. Antiaging Effects of Compound 9 Diminished in Uth1, Skn7, Sod1, and Sod2 Mutations with K6001 Background. To investigate the role of these genes in the antiaging activity of compound 9, we used the mutants of uth1, skn7, sod1, and sod2. As shown in Figure 6, compound 9 at 3 μM did not affect the replicative lifespan of uth1 (Figure 6(a)) or skn7 mutants (Figure 6(b)), neither of sod1 (Figure 6(c)) or sod2 mutants (Figure 6(d)). These results were further indicated that these four genes were involved in the mechanism of action of compound 9.

Conclusions
A novel cucurbitane-type triterpenoid and nine known compounds were isolated and identified from the fruits of M. charantia. All the compounds showed antiaging effect in The average lifespan of Δskn7 in the control group was 9.88 ± 0.41 and compound 9 at 3 μM, 10.40 ± 0.53. (c) The average lifespan of Δsod1 in the control group was 6.55 ± 0.32 and compound 9 at 3 μM, 6.65 ± 0.34. (d) The average lifespan of Δsod2 in the control group was 6.28 ± 0.25 and compound 9 at 3 μM, 6.50 ± 0.25 ( * * P < 0 01 and * * * P < 0 001).
yeast. The antiaging activities of these cucurbitane-type triterpenoids depended on their antioxidative ability and the regulation of the UTH1, SKN7, SOD1, and SOD2 yeast genes. Apart from being one of the most well-known vegetables and frequently used as a traditional medicine because of its health benefits, M. charantia has potential as an antiaging functional food.